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BBA - Proteins and Proteomics (v.1784, #9)

Editorial Board (pp. ii).

In Memoriam by Yuji Goto (pp. 1127-1129).

Seprase: An overview of an important matrix serine protease by Pamela O'Brien; Brendan F. O'Connor (pp. 1130-1145).

Seprase or Fibroblast Activation Protein (FAP) is an integral membrane serine peptidase, which has been shown to have gelatinase activity. Seprase has a dual function in tumour progression. The proteolytic activity of Seprase has been shown to promote cell invasiveness towards the ECM and also to support tumour growth and proliferation. Seprase appears to act as a proteolytically active 170-kDa dimer, consisting of two 97-kDa subunits. It is a member of the group type II integral serine proteases, which includes dipeptidyl peptidase IV (DPPIV/CD26) and related type II transmembrane prolyl serine peptidases, which exert their mechanisms of action on the cell surface. DPPIV and Seprase exhibit multiple functions due to their abilities to form complexes with each other and to interact with other membrane-associated molecules. Localisation of these protease complexes at cell surface protrusions, called invadopodia, may have a prominent role in processing soluble factors and in the degradation of extracellular matrix components that are essential to the cellular migration and matrix invasion that occur during tumour invasion, metastasis and angiogenesis.

Keywords: Abbreviations; AEBSF; 4-(2-aminoethyl)-benzenesulfonyl fluoride; AFC; 7-amino-4-trifluoromethylcoumarin; AMC; 7-amino-4-methylcoumarin; APSF; 4-amidino phenylsulfonyl fluoride; DABCYL; 4-(4-dimethylaminophenylazo)benzoyl; DFP; Diisopropyl fluorophosphates; DPPIV; Dipeptidyl peptidase IV; DTT; Dithiothreitol; ECM; Extracellular matrix; EDANS; 5-[(2-aminoethyl)amino]-naphthalene-1-sulfonic acid; EDTA; Ethylenediaminetetra acetic acid; FGF-2; Fibroblast growth factor-2; GBase; Guanidinobutyrase; MMP; Matrix metallo-proteinase; mAb; monoclonal antibody; NEM; N; -ethylmaleimide; PMSF; Phenylmethylsulfonyl fluoride; POP; Prolyl oligopeptidase; SIMP; Serine Integral Membrane Protein; uPA; Urokinase plasminogen activatorSeprase; Fibroblast Activation Protein α; Serine peptidase; Antiplasmin Cleaving Enzyme; Serine integral membrane protein

Protein dynamics explain the allosteric behaviors of hemoglobin by Takashi Yonetani; Monique Laberge (pp. 1146-1158).

Bohr, Hasselbalch, and Krogh discovered homotropic and heterotropic allosteric behaviors of hemoglobin (Hb) in 1903/1904. A chronological description since then of selected principal models of the allosteric mechanism of Hb, such as the Adair scheme, the MWC two-state concerted model, the KNF induced-fit sequential model, the Perutz stereochemical model, the tertiary two-state model, and the global allostery model (an expanded MWC models), is concisely presented, followed by analysis and discussion of their limitations and deficiencies. The determination of X-ray crystallographic structures of deoxy- and ligated-Hb and the structure-based stereochemical model by Perutz are an epoch-making event in this history. However, his assignment of low-affinity deoxy- and high-affinity oxy-quaternary structures of Hb to the T- and R-states, respectively, though apparently reasonable, and as well as his hypothesis that the T-/R-quaternary structural transition regulates the oxygen-affinity, have created confusions and side-tracked studies of Hb on the structure–function relationship. The differences in static molecular structures of Hb between T(deoxy)- and R(oxy)-quaternary states reported in detail by Perutz and others are ligation-linked structural changes, but not related to the control/regulation of the oxygen-affinity. The oxygen-affinity ( K_T and K_R) of Hb has been shown to be regulated by the heterotropic effector-linked tertiary structural changes without involving the T/R-quaternary changes. However, a recent high-resolution crystallographic analysis of Hb with different oxygen-affinities shows that static molecular structures of Hb determined by crystallography can neither identify the nature of the T(low-affinity) functional state nor decipher the mechanism by which Hb stores free energy in the T(low-affinity) functional state. Molecular dynamics simulations show that fluctuations of helices of oxy-Hb are increased upon de-oxygenation and/or binding 2,3-biphosphoglycerate. These are known to lower the oxygen-affinity of Hb. It is proposed that the coordination mode of the heme Fe with proximal and distal His is modulated by these helical fluctuations, resulting in the modulation of the oxygen-affinity of Hb. Therefore, it is proposed that the oxygen-affinity of Hb is regulated by pentanary (the 5th-order time-dependent or dynamic) tertiary structural changes rather than the T-/R-quaternary structural transitions in Hb. Homotropic and heterotropic allosteric effects of Hb are oxygen- and effector-linked, conformational entropy-driven entropy-enthalpy compensation phenomena and not much to do with static structural changes. The dynamic allostery model, which integrates these observations, provides the structural basis for the global allostery model (an expanded MWC model).

Keywords: Abbreviations; Hb; hemoglobin; MD; molecular dynamics; BPG; 2,3-biphosphoglycerate; MWC; Monod, Wyman and Changeux; KNF; Koshland, Nemethy and FilmerHemoglobin (Hb); Oxygen-binding; Allostery; Cooperativity; Quaternary structure; Molecular dynamics

High mobility group proteins and their post-translational modifications by Qingchun Zhang; Yinsheng Wang (pp. 1159-1166).

The high mobility group (HMG) proteins, including HMGA, HMGB and HMGN, are abundant and ubiquitous nuclear proteins that bind to DNA, nucleosome and other multi-protein complexes in a dynamic and reversible fashion to regulate DNA processing in the context of chromatin. All HMG proteins, like histone proteins, are subjected to extensive post-translational modifications (PTMs), such as lysine acetylation, arginine/lysine methylation and serine/threonine phosphorylation, to modulate their interactions with DNA and other proteins. There is a growing appreciation for the complex relationship between the PTMs of HMG proteins and their diverse biological activities. Here, we reviewed the identified covalent modifications of HMG proteins, and highlighted how these PTMs affect the functions of HMG proteins in a variety of cellular processes.

Keywords: High mobility group protein; Post-translational modification; Mass spectrometry

Comparative studies of wild type Escherichia coli 5-enolpyruvylshikimate 3-phosphate synthase with three glyphosate-insensitive mutated forms: Activity, stability and structural characterization by Karimeh Haghani; Ali Hatef Salmanian; Bijan Ranjbar; Kobra Zakikhan-Alang; Khosro Khajeh (pp. 1167-1175).

5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase is an essential enzyme of the shikimate pathway and is the target for the herbicide, glyphosate. Several glyphosate-insensitive forms of Escherichia coli EPSP synthase had been reported in the literatures. In the present study the function and structure of wild type enzyme and three different mutated variants (G96A, A183T and G96A/A183T) were compared. Results showed that G96A and G96A/A183T variants are insensitive to glyphosate but display a 31- and 8-fold lower affinity for phosphoenolpyruvate (PEP) as substrate, respectively. In addition, chemical stability of the enzyme variants against Gdn-HCl revealed more stability of the wild type and G96A variant when compared to the G96A/A183T and A183T variants. Comparison of the enzymes containing Ala183Thr replacement with the wild type showed a lower resistance to digestion by the proteases. Moreover, with respect to fluorescence quenching by acrylamide, A183T and G96A/A183T variants were characterized by a higher structural flexibility and more exposure of tryptophan residues to the solvent. In addition, based on the results of circular dichroism and intrinsic fluorescence studies, these two variants represent a significant decrease of secondary structures and changes in the tertiary structure as compared to the wild type and the G96A variant.

Keywords: Abbreviations; EPSP synthase; 5-enolpyruvylshikimate 3-phosphate synthase; CD; circular dichroism5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase; Glyphosate; Stability; Site-directed mutagenesis; Circular dichroism; Fluorescence studies

The conserved N-terminal helix of acylpeptide hydrolase from archaeon Aeropyrum pernix K1 is important for its hyperthermophilic activity by Zuoming Zhang; Baisong Zheng; Yanping Wang; Yiqian Chen; Giuseppe Manco; Yan Feng (pp. 1176-1183).

The acylpeptide hydrolases from hyperthermophilic archaeon Aeropyrum pernix K1 has a short conserved N-terminal helix in its family. The role of this N-terminal helix in the function of the hyperthermophilic enzyme, however, is unknown. Here, we investigated this question by protein engineering and biophysical methods. We found that a mutant (ΔN21) with the N-terminal helix deleted is no longer functional at the optimum temperature for WT enzyme (95 °C), required for the survival of Aeropyrum pernix K1. Instead, ΔN21 has the optimum activity at ∼77 °C, with higher activities than the WT enzyme below this temperature. ΔN21 is less stable than the WT enzyme and started unfolding at ∼77 °C, indicating that the loss of the enzymatic activity of ΔN21 at higher temperature is due to its low thermodynamic stability. In addition, we found that the salt bridges formed between the N-terminal helix and the catalytic domain of the enzyme play only a minor role in stabilizing the enzyme, suggesting that hydrophobic interactions mainly contribute to the stabilization. Since the N-terminal helix is conserved in this family of enzymes, our results suggest that the N-terminal helix is likely to play an important role for stabilizing all other enzymes in this family.

Keywords: Abbreviations; APH; acylpeptide hydrolase; POP; prolyl oligopeptidase; OB; oligopeptidase B; apAPH; APH protein from; Aeropyrum pernix; K1; E. coli; Escherichia coli; p; NA; p; -nitroanilide; p; NPC8; p; -nitrophenyl caprylate; WT; wild type protein; DSC; differential scanning calorimetry; GdmCl; guanidinium chloride; FIRST; Floppy Inclusion and Rigid Substructure TopologyAcylpeptide hydrolase; Prolyl oligopeptidase family; N-terminal truncation; Protein stability; Molecular dynamics simulation

Proteomic investigation of taxol and taxotere resistance and invasiveness in a squamous lung carcinoma cell line by L. Murphy; M. Henry; P. Meleady; M. Clynes; J. Keenan (pp. 1184-1191).

Pulse selections on a chemotherapy naive squamous lung carcinoma cell line, SKMES-1, with clinically relevant concentrations of taxanes (taxol or taxotere) resulted in the development of a stable taxotere-resistant variant, SKMES-1-Taxotere and an unstable taxol-resistant variant, SKMES-1-Taxol. Both variants exhibited increased invasiveness in vitro. The unstable nature of SKMES-1-Taxol facilitated looking at factors involved in loss of taxol resistance and increased invasion. The taxotere and taxol-resistant cell lines were 5.9-fold and 12.5-fold resistant to taxotere and taxol respectively. Alterations in expression of/or point mutations in tubulin, the main target of taxanes, is a major mechanism or resistance. However, alterations in expression of beta tubulin were not consistent in the taxane-selected variants. Cross-resistance to adriamycin, vincristine and etoposide (VP-16) was consistent with overexpression of P-glycoprotein (P-gp). However, P-gp alone is not sufficient to confer the complete multiple drug resistance phenotype as all cell lines exhibited cross-resistance to 5-Fluorouracil (5-FU) and more than one mechanism has been linked to taxane resistance. There was no correlation between the fall of taxol resistance in SKMES-1-Taxol and P-gp expression indicating the loss in resistance to be independent of P-gp expression. Furthermore, resistance to the other drugs was not unstable in SKMES-1-Taxol suggesting some parallel mechanisms of resistance. Two-dimensional electrophoresis coupled with matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry was used to identify alterations in expression of specific proteins associated with taxane resistance. A large number of differentially regulated proteins were identified in the resistant and invasive variants affecting cellular processes including stress response, protein turnover and cytoskeleton proteins.

Keywords: Abbreviations; VP-16; etopside; P-gp; P-glycoprotein; 5-FU; 5-fluorouracil; MALDI-TOF; matrix assisted laser desorption ionisation-time of flight; PBS; phosphate buffered saline; ATCC; American type culture collection; 2D-DIGE; two dimensional-difference in gel electrophoresis; SDS-PAGE; sodium dodecyl sulphate polyacrylamide gel electrophoresis; BVA; biological variation analysis; BSA; bovine serum albumin; 2-DE; 2-dimensional electrophoresis; HSP; heat shock protein; ROS; reactive oxygen species; MMP; matrix metalloproteinase; VCR; vincristine; GST-π; glutathione-; S; -transferase-π; VCP; valosin containing protein; PHB; prohibitinDrug-resistance; Lung cancer; Taxol; Taxotere; Proteomics

Differential rate constants of racemization of aspartyl and asparaginyl residues in human alpha A-crystallin mutants by Tooru Nakamura; Miyo Sakai; Yutaka Sadakane; Tatsuya Haga; Yuji Goto; Tadatoshi Kinouchi; Takeshi Saito; Noriko Fujii (pp. 1192-1199).

Asp58 and Asp151 in alpha A-crystallin of human eye lenses become highly inverted and isomerized tod-beta-Asp residues with age. Racemization was previously shown to proceed rapidly when the residue on the carboxyl side of the Asp residue is small. Asn was also demonstrated to be more susceptible to racemization than Asp in protein. In this study, the changes of rate constants for racemization at Asp58 and Asp151 and at Asn58 and Asn151 were investigated using D58N, S59T, D151N and A152V mutants obtained through site-directed mutagenesis. The rate constant of racemization at Asn151 in D151N was found to be 1.5 times more rapid than Asp151 in the wild-type. For A152V, the rate constant at Asp151 was 1/4 that of the wild-type. There were no significant differences in the rate constants of racemization for both Asp58 and Asn58 residues. The aggregate size of D58N, S59T and D151N mutants increased or increased in polydispersity and their chaperone activities decreased. The size and chaperone activity of A152V was unchanged. These results suggest that structures close to Asp58 and Asp151 residues in the protein affect the rate constant of Asp racemization and the size and chaperone function of alpha A-crystallin.

Keywords: Recombinant human alpha A-crystallin; Racemization; D-aspartyl residue; Lens; Mutant

Fourier transform coupled to tryptophan-scanning mutagenesis: Lessons from its application to the prediction of secondary structure in the acetylcholine receptor lipid-exposed transmembrane domains by José David Otero-Cruz; Nunez David Abner Torres-Núñez; Baez-Pagan Carlos Alberto Báez-Pagán; José Antonio Lasalde-Dominicci (pp. 1200-1207).

Although Fourier transform (FT) and tryptophan-scanning mutagenesis (TrpScanM) have been extremely useful for predicting secondary structures of membrane proteins, they are deemed to be low-resolution techniques. Herein, we describe the combined use of FT and TrpScanM (FT-TrpScanM) as a more reliable approach for the prediction of secondary structure. Five TrpScanM studies of the acetylcholine receptor lipid-exposed transmembrane domains (LETMDs) were revisited and analyzed by FT-TrpScanM. FT analysis of the raw data from the aforementioned TrpScanM studies supports and validates the conclusions derived from their tryptophan-periodicity profiles. Furthermore, by FT-TrpScanM, we were able to determine the minimum number of consecutive tryptophan substitutions necessary for more robust prediction of α-helical secondary structures and evaluate the quality of structure predictions by α-helical character curves. Finally, this study encourages future utilization of FT-TrpScanM to more reliably predict secondary structures of the membrane protein LETMDs.

Keywords: Abbreviations; AChR; acetylcholine (ACh) receptor; FT; Fourier transform; FT-TrpScanM; Fourier transform coupled to tryptophan-scanning mutagenesis; LETMDs; lipid-exposed transmembrane domains; TrpPPs; tryptophan-periodicity profiles; TrpScanM; tryptophan-scanning mutagenesisTryptophan-scanning mutagenesis; Fourier transform; Ligand-gated ion channel; Lipid-exposed transmembrane domain; Secondary structure; Two-electrode voltage clamp

Structural characterisation of PinA WW domain and a comparison with other Group IV WW domains, Pin1 and Ess1 by Chai Ann Ng; Yusuke Kato; Masaru Tanokura; Robert T.C. Brownlee (pp. 1208-1214).

The NMR solution structure of the PinA WW domain from Aspergillus nidulans is presented. The backbone of the PinA WW domain is composed of a triple-stranded anti-parallel β-sheet and an α-helix similar to Ess1 and Pin1 without the α-helix linker. Large RMS deviations in Loop I were observed both from the NMR structures and molecular dynamics simulation suggest that the Loop I of PinA WW domain is flexible and solvent accessible, thus enabling it to bind the pS/pT-P motif. The WW domain in this structure are stabilised by a hydrophobic core. It is shown that the linker flexibility of PinA is restricted because of an α-helical structure in the linker region. The combination of NMR structural data and detailed Molecular Dynamics simulations enables a comprehensive structural and dynamic understanding of this protein.

Keywords: Abbreviations; MD; molecular dynamics; pS/pT-P motif; phospho-Serine/Threonine-Proline motifs; PPIase; petidyl-prolyl; cis; –; trans; -isomerase; p patch; phosphate binding patch; Pin1 WW domain; the WW domain of Pin1; XP groove; Xxx-Pro binding groove; PinA WW domain; the WW domain of PinA; NOE; nuclear Overhauser effect; DSS; internal 2,2-dimethyl-2-silapentane-5-sulfate sodium salt; CSI; ¹³; C chemical shift index; NPT; constant pressure dynamicsWW domain; Structure comparison; Protein structure; Molecular dynamics; NMR

Disulfide bond within µ-calpain active site inhibits activity and autolysis by René Lametsch; Steven Lonergan; Elisabeth Huff-Lonergan (pp. 1215-1221).

Oxidative processes have the ability to influence µ-calpain activity. In the present study the influence of oxidation on activity and autolysis of µ-calpain was examined. Furthermore, LC-MS/MS analysis was employed to identify and characterize protein modifications caused by oxidation. The results revealed that the activity of µ-calpain is diminished by oxidation with H₂O₂ in a reversible manner involving cysteine and that the rate of autolysis of µ-calpain concomitantly slowed. The LC-MS/MS analysis of the oxidized µ-calpain revealed that the amino acid residues 105–133 contained a disulfide bond between Cys(108) and Cys(115). The finding that the active site cysteine in µ-calpain is able to form a disulfide bond has, to our knowledge, not been reported before. This could be part of a unique oxidation mechanism for µ-calpain. The results also showed that the formation of the disulfide bond is limited in the control (no oxidant added), and further limited in a concentration-dependent manner when β-mercaptoethanol is added. However, the disulfide bond is still present to some extent in all conditions indicating that the active site cysteine is potentially highly susceptible to the formation of this intramolecular disulfide bond.

Keywords: Abbreviations; LC-MS/MS; liquid chromatography coupled with mass spectrometry; Cys; cysteine; MCE; β-mercaptoethanol; ROS; reactive oxygen species; Tris; 2-amino-2-(hydroxymethyl)-1,3-propanediol; EDTA; ethylenediaminetetraacetic acid; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis; NEM; N; -Ethylmaleimide; TOF; time of flightµ-Calpain; Oxidation; LC-MS; Cysteine modification; Disulfide bond

Functional guanine–arginine interaction between tRNA^Pro and prolyl-tRNA synthetase that couples binding and catalysis by Brian Burke; Songon An; Karin Musier-Forsyth (pp. 1222-1225).

Aminoacyl-tRNA synthetases catalyze the attachment of specific amino acids to their cognate tRNAs. Specific aminoacylation is dictated by a set of recognition elements that mark tRNA molecules as substrates for particular synthetases. Escherichia coli prolyl-tRNA synthetase (ProRS) has previously been shown to recognize specific bases of tRNA^Pro in both the anticodon domain, which mediate initial complex formation, and in the acceptor stem, which is proximal to the site of catalysis. In this work, we unambiguously define the molecular interaction between E. coli ProRS and the acceptor stem of cognate tRNA^Pro. Oxidative cross-linking studies using 2'-deoxy-8-oxo-7,8-dihydroguanosine-containing proline tRNAs identify a direct interaction between a critical arginine residue (R144) in the active site of E. coli ProRS and the G72 residue in the acceptor stem of tRNA^Pro. Assays conducted with motif 2 loop variants and tRNA mutants wherein specific atomic groups of G72 were deleted, are consistent with a functionally important hydrogen-bonding network between R144 and the major groove of G72. These results taken together with previous studies suggest that breaking this key contact uncouples the allosteric interaction between the anticodon domain and the aminoacylation active site, providing new insights into the communication network that governs the synthetase–tRNA interaction.

Keywords: Aminoacyl-tRNA synthetases; Prolyl-tRNA synthetase; Oxidative cross-linking; 8-oxo-guanosine; RNA–protein interactions; Coupling network

Mutant forms of the extracellular domain of the human acetylcholine receptor γ-subunit with improved solubility and enhanced antigenicity by Kalliopi Bitzopoulou; Kalliopi Kostelidou; Konstantinos Poulas; Socrates J. Tzartos (pp. 1226-1233).

The muscle nicotinic acetylcholine receptor (AChR) is the prototype of the ligand-gated ion channels (or Cys-loop receptors), formed by 5 homologous subunits (α₂βγδ or α₂βγɛ), and is the major autoantigen in the autoimmune disease, myasthenia gravis. Previously, we expressed the wild-type extracellular domain (ECD) of the γ-subunit (γECD) of the AChR in yeast Pichia pastoris at 0.3–0.8 mg/L, in soluble but microaggregate form, to use as starting material for structural and antigenicity studies. To optimize these characteristics, we constructed and characterized four γECD variants: (a) mutants-1 (γC61S) and -2 (γC106S-C115S), where the non-conserved Cys of γECD were replaced by serines, (b) mutant-3 (γCysLoop), where the γ Cys-loop region was substituted by the cognate region of the acetylcholine binding protein (AChBP) and (c) mutant-4 (γCysLoop-C106S-C115S), where both the C106S-C115S and Cys-loop mutations were combined. None of mutants-1 and -2 displayed any improvement, while mutant-3 and -4 were mostly in dimeric form and expressed at much higher levels (2.5 mg/L and 3.5 mg/L respectively). All four mutants and wild-type γECD were recognized by sera from myasthenic patients, but mutants-3 and -4 exhibited higher efficiency, compared to wild-type or mutants-1 and -2. These results suggest that the substitution of the Cys-loop region of any AChR ECD with the AChBP counterpart leads to AChR ECD of improved conformation, more suitable for structural and therapeutic studies.

Keywords: Abbreviations; AChBP; acetylcholine binding protein; AChR; nicotinic acetylcholine receptor; γECD; amino acids 1-218 of the human wild-type AChR γ-subunit; γC61S; γECD where the Cys at position 61 is replaced by serine; γC106S-C115S; γECD where the Cys at positions 106 and 115 are replaced by serines; γCysLoop; γECD where the Cys-loop of the γECD is replaced by the Cys-loop of the AChBP; γCysLoop-C106S-C115S; γECD where Cys at positions 106 and 115 are replaced by serines and the Cys-loop is replaced by the Cys-loop of the AChBP; DLS; dynamic light scattering; ECD; extracellular domain; LGIC; ligand-gated ion channels; MG; myasthenia gravis; PBS; phosphate-buffered saline; RDB; regeneration dextrose baseAcetylcholine receptor; Protein expression; Extracellular domain; Myasthenia gravis; Immunoadsorption

Bovine viral diarrhea viruses differentially alter the expression of the protein kinases and related proteins affecting the development of infection and anti-viral mechanisms in bovine monocytes by George V. Pinchuk; Sang-Ryul Lee; Bindu Nanduri; Kelly L. Honsinger; John V. Stokes; Lesya M. Pinchuk (pp. 1234-1247).

Using a proteomics approach, we evaluated the effect of cytopathic (cp), and non-cytopathic (ncp) bovine viral diarrhea viruses (BVDV) on the expression of protein kinases and related proteins in bovine monocytes. Proteins were isolated from membrane and cytosolic fractions with the differential detergent fractionation (DDF) method and identified with 2D-LC ESI MS². Of approximately 10,000 proteins identified, 378 proteins had homology with known protein kinases or related proteins. Eighteen proteins involved in cell differentiation and activation, migration, anti-viral mechanisms (interferon/apoptosis), biosynthesis, sugar metabolism and oncogenic transformation were significantly altered in BVDV-infected monocytes compared to the uninfected controls. Six proteins, mostly related to cell migration, anti-viral mechanisms, sugar metabolism and possibly tumor resistance were differentially expressed between the ncp and cp BVDV-infected monocytes. Particularly, the expression of the receptor of activated C kinase (RACK), of pyridoxal kinase (PK), diacyglycerol kinase (DGK) and Brutons tyrosine kinase (BTK) was decreased in monocytes infected with cp BVDV compared to ncp BVDV, possibly contributing to the cytopathic effect of the virus. This and other findings are discussed in view of the possible role the identified proteins play in the development of viral infection and oncogenic transformation of cells.

Keywords: Abbreviations; cp; cytopathic; ncp; non-cytopathic; BVDV; bovine viral diarrhea virus; DDF; differential detergent fractionation; 2D-LC ESI MS; ²; two-dimensional liquid chromatography electrospray ionization tandem mass spectrometry; u-PAR; urokinase-type plasminogen activator receptor; MARCKS; myristoilated alanine-rich C kinase substrate; NDKB; nucleoside diphosphate kinase B; ROK1; Rho-associated protein kinase 1; HK3; hexokinase type 3; DCK2; doublecortin kinase 2; KSR2; kinase suppressor of Ras 2; RACK; receptor of activated C kinase; GK1; galactokinase 1; PK; pyridoxal kinase; STK; spleen tyrosine kinase; PK-M1/M2; pyruvate kinase isozymes M1 and M2; fgr; kinase; proto-oncogene tyrosine kinase FGR; MAPKKK; mitogen-activated protein tyrosine kinase kinase kinase; PKC; protein kinase C; TYRO protein; TYRO protein tyrosine kinase-binding protein precursor; DGK; diacyglycerol kinase; BTK; Brutons tyrosine kinase; PFK; 6-phosphofructo-2-kinase/fructose-2,6-biphosphate kinaseMonocyte; Protein kinase; Anti-viral mechanism; BVDV; DDF; MudPIT; 2D-LC ESI MS; ²; X; corr

Physical, kinetic and spectrophotometric studies of a NAD(P)-dependent benzaldehyde dehydrogenase from Pseudomonas putida ATCC 12633 by Catherine K. Yeung; Alejandra Yep; George L. Kenyon; Michael J. McLeish (pp. 1248-1255).

The mandelate pathway of Pseudomonas putida ATCC 12633 comprises five enzymes and catalyzes the conversion of R- and S-mandelamide to benzoic acid which subsequently enters the β-ketoadipate pathway. Although the first four enzymes have been extensively characterized the terminal enzyme, a NAD(P)⁺-dependent benzaldehyde dehydrogenase (BADH), remains largely undescribed. Here we report that BADH is a dimer in solution, and that DTT is necessary both to maintain the activity of BADH and to prevent oligimerization of the enzyme. Site-directed mutagenesis confirms that Cys249 is the catalytic cysteine and identifies Cys140 as the cysteine likely to be involved in inter-monomer disulfide formation. BADH can utilize a range of aromatic substrates and will also operate efficiently with cyclohexanal as well as medium-chain aliphatic aldehydes. The log V and log V/ K pH-rate profiles for benzaldehyde with either NAD⁺ or NADP⁺ as the coenzyme are both bell-shaped. The p K_a values on the ascending limb range from 6.2 to 7.1 while those on the descending limb range from 9.6 to 9.9. A spectrophotometric approach was used to show that the p K_a of Cys249 was 8.4, i.e., Cys249 is not responsible for the p K_as observed in the pH-rate profiles.

Keywords: Mandelate pathway; Gene cloning; Aldehyde dehydrogenase; pH dependence; Catalytic thiol; p; K; _a

A novel serralysin metalloprotease from Deinococcus radiodurans by Bhakti Basu; Shree Kumar Apte (pp. 1256-1264).

A hypothetical protein (DR2310) from the radiation resistant organism Deinococcus radiodurans harbors highly conserved Zn⁺²-binding (HEXXH) domain and Met-turn (SVMSY), characteristic of the serralysin family of secreted metalloproteases from Gram negative bacteria. Deletion mutagenesis of DR2310 confirmed that the ORF is expressed in Deinococcus radiodurans as a secreted protease of 85 kDa. Biochemical analysis revealed DR2310 to be a Ca⁺² and Zn⁺²-requiring metalloprotease. Unique features such as a long N-terminus, replacement of the highly conserved C-terminal glycine rich Ca⁺²-binding repeats with a single N-terminal aspartate rich eukaryotic thrombospondin type-3 Ca⁺²-binding repeat and absence of C-terminal secretion signals make it a novel member of serralysin family. This is the first report of a functional serralysin family metalloprotease from a Gram positive organism.

Keywords: Abbreviations; MES; 2-(; N; -morpholino)ethanesulfonic acid; PIPES; Piperazine-1,4-bis(2-ethanesulfonic acid; HEPES; N-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid; 1,10-Phe; 1,10-phenanthroline; EDTA; Ethylene diamine tetra acetic acid; EGTA; Ethylene glycol-bis(beta-aminoethyl ether)-; N; ,; N; ,; N; ′,; N; ′-tetra acetic acid; PMSF; Phenyl Methyl Sulfonyl Fluoride; E-64; (2S,3S)-3-(; N; -{(S)-1-[; N; -(4-guanidinobutyl)carbamoyl]3-methylbutyl}carbamoyl) oxirane-2-carboxylic acid; Pep; Pepstatin; Ca; ⁺²; Calcium; Co; ⁺²; Cobalt; Mg; ⁺²; Magnesium; Mn; ⁺²; Manganese; Zn; ⁺²; Zinc Deinococcus; Serralysin-like extracellular metalloprotease; Zn; ⁺²; /Ca; ⁺²; -dependency

Small angle X-ray scattering study of calreticulin reveals conformational plasticity by Norgaard Toft Katrine Nørgaard Toft; Nanna Larsen; Steen Jorgensen Flemming Steen Jørgensen; Hojrup Peter Højrup; Gunnar Houen; Bente Vestergaard (pp. 1265-1270).

Calreticulin plays a central role in vital cell processes such as protein folding, Ca²⁺ homeostasis and immunogenicity. Even so, only limited three-dimensional structural information is presently available. We present a series of Small-Angle X-ray Scattering data on human placenta calreticulin. The data from the calreticulin monomer reveal the shape of calreticulin in solution: The previously structurally un-described C-terminal is seen as a globular domain, and the P-domain β-hairpin extends from the N-domain in a spiral like conformation. In the calreticulin solution dimer, the N-, C-, and P-domains are easily identified, and the P-domain is in an extended conformation connecting to the second calreticulin molecule. The SAXS solution data enables the construction of a medium-resolution model of calreticulin. In the light of the unresolved chaperone mechanism of calreticulin and calnexin, we discuss the functional consequences of the conformational plasticity of the calreticulin P-domain.

Keywords: Calreticulin; SAXS; Protein folding; Chaperone; Endoplasmic reticulum

Interaction of the globular domain of human C1q with Salmonella typhimurium lipopolysaccharide by Lubka T. Roumenina; Krustyo T. Popov; Svetlana V. Bureeva; Mihaela Kojouharova; Mihaela Gadjeva; Shweta Rabheru; Roshni Thakrar; Alexander Kaplun; Uday Kishore (pp. 1271-1276).

Gram-negative bacteria can bind complement protein C1q in an antibody-independent manner and activate classical pathway via their lipopolysaccharides (LPS). Earlier studies have implicated the collagen-like region of human C1q in binding LPS. In recent years, a number of C1q target molecules, previously considered to interact with collagen-like region of C1q, have been shown to bind via the globular domain (gC1q). Here we report, using recombinant forms of the globular head regions of C1q A, B and C chains, that LPS derived from Salmonella typhimurium interact specifically with the B-chain of the gC1q domain in a calcium-dependent manner. LPS and IgG-binding sites on the gC1q domain appear to be overlapping and this interaction can be inhibited by a synthetic C1q inhibitor, suggesting common interacting mechanisms.

Keywords: Abbreviations; B2S; betulin disulfate; gC1q; the globular domain of human C1q; CLR; collagen-like region of C1q; CRP; C-reactive protein; ghA; ghB, ghC, the recombinant forms of the globular regions of C1q A, B and C chains, respectively; F2S; 9,9-bis(4′hydroxyphenyl)fluorene disulfate; KDO; 2-keto-3-deoxyoctonoic acid; LPS; lipopolysaccharides; MBP; maltose-binding protein; pIgG; pooled human IgG; PBS; phosphate-buffered saline; PTX3; long prototypic pentraxin 3; PBST; phosphate-buffered saline containing 0.05% Tween-20; HRP; horseradish peroxidaseC1q; Salmonella lipopolysaccharide; IgG; Ca; ²⁺; Recognition

Purification and characterization of paralytic shellfish toxin-transforming enzyme, sulfocarbamoylase I, from the Japanese bivalve Peronidia venulosa by Yuko Cho; Noriyuki Ogawa; Miyako Takahashi; Hsi-Pin Lin; Yasukatsu Oshima (pp. 1277-1285).

The Japanese bivalve Peronidia venulosa contains paralytic shellfish toxin (PST)-transforming enzymes that convert the weakly toxic C-toxins to the more potent decarbamoyl toxins. The enzyme was purified 154-fold with a yield of 0.26% and was named sulfocarbamoylase I. It was found to be a protein with an estimated molecular weight of 300 kDa by gel filtration column chromatography. Observation of a single band equivalent to 150 kDa on SDS-PAGE with or without reducing agents suggested it to be a homodimer with ionically bound subunits. The enzyme catalyzes the hydrolysis of the carboxyl bond in the N-sulfocarbamoyl moiety of PSP-toxins. The sulfonyl moiety in the carbamoyl side chain of substrates is essential for enzyme recognition. The N-terminal amino acid sequences of nine tryptic peptides were determined by the Edman degradation method. In a database search using the BLAST program, no protein that shows remarkable homology was retrieved. Several characteristics of the enzyme were also compared with those of another PST-transforming enzyme, carbamoylase I, which was previously isolated from the Japanese clam Mactra chinensis.

Keywords: Peronidia venulosa; Crystalline style; Sulfocarbamoylase; Paralytic shellfish toxins; Saxitoxin

Mechanism of thermal aggregation of yeast alcohol dehydrogenase I by Kira A. Markossian; Nikolay V. Golub; Helen A. Khanova; Dmitrii I. Levitsky; Nikolay B. Poliansky; Konstantin O. Muranov; Boris I. Kurganov (pp. 1286-1293).

Kinetics of thermal aggregation of yeast alcohol dehydrogenase I (yADH) have been studied using dynamic light scattering at a fixed temperature (56 °C) and under the conditions where the temperature was elevated at a constant rate (1 K/min). The initial parts of the dependences of the hydrodynamic radius on time (or temperature) follow the exponential law. At rather high values of time splitting of the population of aggregates into two components occurs. It is assumed that such peculiarities of the kinetics of thermal aggregation of yADH are due to the presence of a sequence –YSGVCHTDLHAWHGDWPLPVK– in the polypeptide chain possessing chaperone-like activity. Thermodynamic parameters for thermal denaturation of yADH have been calculated from the differential scanning calorimetry data.

Keywords: Yeast alcohol dehydrogenase I; Denaturation; Aggregation; Intramolecular chaperone; Dynamic light scattering

The action of MBL-associated serine protease 1 (MASP1) on factor XIII and fibrinogen by Anders Krarup; Krishana C. Gulla; Peter Gal Péter Gál; Krishnan Hajela; Robert B. Sim (pp. 1294-1300).

The complement system is an important recognition and effector mechanism of the innate immune system that upon activation leads to the elimination of foreign bodies. It can be activated through three pathways of which the lectin pathway is one. The lectin pathway relies on the binding of mannan-binding lectin (MBL) or the ficolins and the subsequent activation of the MBL-associated serine proteases (MASPs), namely, MASP1, 2 and 3 which all form complexes with both MBL and the ficolins. Major substrates have only been identified for MASP2 i.e. C4 and C2. For MASP1 only a few protein substrates which are cleaved at a low rate have been identified while none are known for MASP3. Since chromogenic substrate screenings have shown that MASP1 has thrombin-like activity, we wanted to investigate the catalytic potential of MASP1 towards two major proteins involved in the clotting process, fibrinogen and factor XIII, and compare the activity directly with that of thrombin. We found that rMASP1 and thrombin cleave factor XIII A-chain and the fibrinogen β-chain at identical sites, but differ in cleavage of the fibrinogen α-chain. The thrombin turnover rate of factor XIII is approximately 650 times faster than that of rMASP1 at 37°C, pH 7.4. rMASP1 cleavage of fibrinogen leads to the release of the proinflammatory peptide fibrinopeptide B. Thus rMASP1 has similar, but not identical specificity to thrombin and its catalytic activity for factor XIII and fibrinogen cleavage is much lower than that of thrombin. Nevertheless, rMASP1 can drive the formation of cross-linked fibrinogen. Since MASP1 is activated on contact of MBL or the ficolins with microorganisms, fibrinogen and factor XIII may be involved in the elimination of invading pathogens.

Keywords: Complement; Lectin pathway; Coagulation system; MASP; Factor XIII; Fibrinogen

Stability of dystrophin STR fragments in relation to junction helicity by Ahmed Mirza; Nick Menhart (pp. 1301-1309).

Dystrophin is a rod shaped protein consisting of amino- and carboxy-terminal binding domains linked by a large central rod composed of 24 homologous copies of the STR motif and 4 non-homologous regions termed hinges. These hinges are proposed to confer local flexibility; conversely, the tacit implication is that the STR regions away from the hinges are comparatively rigid. This, and the repeating nature of this rod, has contributed to the view that the STR region of the rod is uniform and monolithic. However, we have produced various 2 STR fragments, chosen to have high and low α-helix content at their junctions with each other, and show that they exhibit markedly different stabilities. In contrast to a related protein, spectrin, these differences are not correlated with the calculated helicity, but appear to be an intrinsic property of the motifs themselves. A full understanding of how these properties vary along the length of the rod has implications for the engineering of these rods regions in exon skipping and minidystrophin therapies.

Keywords: Dystrophin; Spectrin-type-repeat; Domain; Stability

Role of the 88–97 loop in plasminogen activation by streptokinase probed through site-specific mutagenesis by Suman Yadav; Manish Datt; Balvinder Singh; Girish Sahni (pp. 1310-1318).

The role of a prominent surface-exposed loop (residues 88–97) in the α domain of streptokinase (SK), in human plasminogen (HPG) activation was explored through its selective mutagenesis and deletion studies. We first made a conformationally constrained derivative of the loop by the substitution of sequences known to possess a strong propensity for β-turn formation. The mutant so formed (termed SK_{88–97-Beta Turn}), when tested for co-factor activity against substrate HPG, after first forming a 1:1 molar complex with human plasmin (HPN), showed a nearly 6-fold decreased co-factor activity compared to the wild-type, native SK. The major catalytic change was observed to be at the k_cat level, with relatively minor changes in K_m values against HPG. Real-time binary interaction (i.e. the 1:1 complexation between SK, or its mutant/s, with HPG), and ternary complexation studies (i.e. the docking of a substrate HPG molecule into the preformed SK–HPG complex) using Surface Plasmon Resonance were done. These studies revealed minor alterations in binary complex formation but the ternary interactions of the substitution and/or deletion mutants were found to be decreased for full-length HPG compared to that for native SK.HPG. In contrast, their ternary interactions with the isolated five-kringle domain unit of plasminogen (K1–5) showed K_d values comparable to that seen with the native SK.HPG complex. Taking into consideration the overall alterations observed in catalytic levels after site-specific mutagenesis and complete loop deletion of the 88–97 loop, on the one hand, and its known position at the SK–HPG interface in the binary complex, suggests the importance of this loop. The present results suggest that the 88–97 loop of the α domain of SK contributes towards catalytic turn-over, even though its individual contribution towards enzyme–substrate affinity per se is minimal.

Keywords: Abbreviations; μPG; microplasminogen; μPN; microplasmin; BSA; bovine serum albumin; CD; circular dichroism; DEAE-Sepharose; diethylaminoethyl-Sepharose; EACA; ɛ-aminocaproic acid; EDC; N′-ethylcarbodiimide; HPG; human plasminogen; HPN; human plasmin; IBs; inclusion bodies; IPTG; isopropyl-1-thio-β-; d; -galactopyranoside; k; _cat; rate of catalysis; K; _d; equilibrium rate constant; K; _m; michaelis-menton constant; k; _off; rate of dissociation; k; _on; rate of association; MDS; molecular dynamics simulations; NHS; N; -hydroxysuccinimide; NPGB; p; -nitrophenyl; p; -guanidinobenzoate; nSK; native-like streptokinase; PAGE; polyacrylamide gel electrophoresis; PBS; phosphate buffered saline; RMSD; root mean square deviation; SAK; staphylokinase; SDS; sodium dodecyl sulphate; SK; streptokinase; SOE-PCR; splicing overlap extension-polymerase chain reaction; SPR; surface plasmon resonance; STI; soybean trypsin inhibitor; tPA; tissue plasminogen activator; UK; UrokinaseStreptokinase; Human plasminogen activation; Plasminogen activator; Protein–protein interaction; Enzyme–substrate interaction

Identification of ERK2-binding domain of EBITEIN1, a novel ERK2-binding protein by Kenji Miura; Junko Imaki (pp. 1319-1325).

We recently cloned a cDNA encoding a novel extracellular signal-regulated kinase 2 (ERK2) binding protein, EBITEIN1, by yeast two-hybrid screening. In this study, we further characterized EBITEIN1. Binding experiments using various deletion mutants identified a 40-amino acid minimal sequence for binding ERK2. Binding experiments using substitution mutants indicated the crucial role of arginine residues in this sequence. Based on empirical and bioinformatic analyses, we propose two domains in EBITEIN1. One is the minimal sequence for binding ERK2 (EB domain) and the other is the EBITEIN1 C-terminal domain (ECT domain). These results might pave the way for further empirical and bioinformatic analyses of EBITEIN1- and ERK2-mediated events.

Keywords: EBITEIN1; Protein–protein interaction; Binding protein; Two-hybrid assay; Binding domain; EB domain; ECT domain; Testis; Bioinformatics

Biochemical and structural characterisation of cutinase mutants in the presence of the anionic surfactant AOT by V. Brissos; E.P. Melo; J.M.G. Martinho; J.M.S. Cabral (pp. 1326-1334).

The reactivity, stability and unfolding of wild-type (WT) Fusarium solani pisi cutinase and L153Q, S54D and T179C variants were studied in the absence and presence of the dioctyl sulfosuccinate sodium salt (AOT) surfactant. In the absence of surfactant the S54D variant catalytic activity is similar to that of the WT cutinase, whereas L153Q and T179C variants show a lower activity. AOT addition induces an activity reduction for WT cutinase and its variants, although for low AOT concentrations a small increase of activity was observed for S54D and T179C. The enzyme deactivation in the presence of 0.5 mM AOT is relatively slow for the S54D and T179C variants when compared to wild-type cutinase and L153Q variant. These results were correlated with secondary and tertiary structure changes assessed by the CD spectrum and fluorescence of the single tryptophan and the six tyrosine residues. The WT cutinase and S54D variant have similar secondary and tertiary structures that differ from those of T179C and L153Q variants. L153Q, S54D and T179C mutations prevent the formation of hydrophobic crevices responsible for the unfolding by anionic surfactants, with the consequent decrease of the AOT–cutinase interactions.

Keywords: Cutinase; AOT; Kinetics; Stability; Protein folding

Distinct interactions between ubiquitin and the SH3 domains involved in immune signaling by Jinho Kang; Sunmi Kang; Hyuk Nam Kwon; Wen He; Sunghyouk Park (pp. 1335-1341).

The SH3 domain is a versatile protein interaction motif that generally recognizes proline rich sequences (PRS). Recently, it has been shown that some SH3 domains in the endocytotic pathway can bind to ubiquitin. Moreover, Phe73 in the SH3 domain has been proposed to be an important determinant of the interaction, as the SH3 domains having Tyr73, either naturally or by mutation, failed to bind. Since SH3 domains are also important in immune receptor signaling, we investigated the interactions between immunologically relevant SH3 domains and ubiquitin. We observed that some of these SH3 domains can also bind to ubiquitin. Interestingly, we found that Nck2-SH3-3 bound to ubiquitin despite its Tyr at residue 73 (Tyr56 in our actual construct), but that CD2BP1-SH3 failed to bind, even though it has Phe at an equivalent position. Through detailed NMR binding studies on SH3 domains with Phes and Tyrs at the 73 position, we found that the two types of SH3 domains exhibit mechanistic differences in ubiquitin binding. We showed that the relative contribution of each binding sub-region in both SH3 domains and ubiquitin is quite different in the two binding modes. Such results raise the possibility that the mechanistic variety of these immunologically relevant SH3 domains might contribute to their functional diversity.

Keywords: Ubiquitin; SH3 domain; Interaction; NMR; Binding mechanism

Acetylation of N-terminal valine of glycine N-methyltransferase affects enzyme inhibition by folate by Zigmund Luka; Lioudmila V. Loukachevitch; Conrad Wagner (pp. 1342-1346).

Native liver glycine N-methyltransferase (GNMT) is N-acetylated while the recombinant enzyme is not. We show here that acetylation of the N-terminal valine affects several kinetic parameters of the enzyme. Glycine N-methyltransferase is a regulatory enzyme mediating the availability of methyl groups by virtue of being inhibited by folate. N-acetylation does not affect the overall structure of the protein and does not affect basal enzyme activity of GNMT. Binding of both the mono- and pentaglutamate forms of 5-methyltetrahydrofolate is the same for the acetylated and non-acetylated forms of the enzyme, however the pentaglutamate form is bound more tightly than the monoglutamate form in both cases. Although binding of the folates is similar for the acetylated and non-acetylated forms of the enzyme, inhibition of enzyme activity differs significantly. The native, N-acetylated form of the enzyme shows 50% inhibition at 1.3 µM concentration of the pentaglutamate while the recombinant non-acetylated form shows 50% inhibition at 590 µM. In addition, the binding of folate results in cooperativity of the substrate S-adenosylmethionine (AdoMet), with a Hill coefficient of 1.5 for 5-methyltetrahydrofolate pentaglutamate.

Keywords: Abbreviations; GNMT; Glycine; N; -methyltransferase; 5-CH; ₃; -THF-Glu; ₁; 5-methyltetrahydrofolate monoglutamate; 5-CH; ₃; -THF-Glu; ₅; 5-methyltetrahydrofolate pentaglutamate; AdoMet; S; -adenosylmethionineGlycine; N; -methyltransferase; Folate; Binding; Inhibition; Cooperativity; N-terminal acetylation

Kinetic and thermodynamic characterization of dUTP hydrolysis by Plasmodium falciparum dUTPase by Indalecio Quesada-Soriano; Isabel Leal; Juan M. Casas-Solvas; Antonio Vargas-Berenguel; Carmen Barón; Luis M. Ruiz-Pérez; Dolores González-Pacanowska; Luis García-Fuentes (pp. 1347-1355).

Deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate and plays an important role in nucleotide metabolism and DNA replication controlling relative cellular levels of dTTP/dUTP, both of which can be incorporated into DNA. Isothermal titration calorimetry has been applied to the determination of the kinetic and thermodynamic parameters of the trimeric Plasmodium falciparum dUTPase, a potential drug target against malaria. The role of divalent ions in binding, and inhibition by different uridine derivatives has been assessed. When dUTP hydrolysis in the presence of EDTA was evaluated, a 105-fold decrease and a 12-fold increase of the k_cat and K_m values, respectively, were observed when compared with the dUTP·Mg²⁺ complex. Calculation of the activation energy, E_a, and the thermodynamic activation parameters showed that the energetic barrier was ~4-fold higher when Mg²⁺ was depleted. Other divalent ions such as Co²⁺ or Mn²⁺ can substitute the physiological cofactor, however the k_cat was significantly reduced compared to dUTP·Mg²⁺. Binding and inhibition by dU, dUMP, dUDP, and α,β-imido-dUTP were analysed by ITC and compared with data obtained by spectrophotometric methods and binding equilibrium studies. Product inhibition ( K_ip dUMP: 99.34 μM) was insignificant yet K_i values for dUDP and α,β-imido-dUTP were in the low micromolar range. The effect of ionic strength on protein stability was also monitored. DSC analysis evidenced a slight increase in the unfolding temperature, T_m, with increasing salt concentrations. Moreover, the thermal unfolding pathway in the presence of salt fits adequately to an irreversible two-state model (N₃→3D).

Keywords: Abbreviations; dUTPase; dUTP pyrophosphatase (EC 3.6.1.23); α; β-imido-dUTP (or dUpNHpp), 2′-deoxyuridine 5′-(α,β-imido)triphosphate; ICP-MS; Inductively Coupled Plasma Mass Spectrometry; ITC; isothermal titration calorimetry; DSC; differential scanning calorimetryKinetic; Calorimetry; dUTPase; Plasmodium falciparum; Thermal denaturation; Binding

Reactions of human liver peroxisomal alanine:glyoxylate aminotransferase with β-chloro-L-alanine andL-cysteine: Spectroscopic and kinetic analysis by Mariarita Bertoldi; Barbara Cellini; Alessandro Paiardini; Riccardo Montioli; Carla Borri Voltattorni (pp. 1356-1362).

In addition to the main transaminase reaction, the pyridoxal 5′-phosphate-dependent enzyme human liver peroxisomal alanine:glyoxylate aminotransferase (AGT) is able to catalyze the α,β-elimination of β-chloro-l-alanine with a catalytic efficiency similar to that of the physiological transaminase reaction withl-alanine. On the other hand, during the reaction of AGT withl-cysteine, changes in the coenzyme forms and analysis of the products reveal the occurrence of both β-elimination and half-transamination ofl-cysteine together with the pyruvate transamination. A mechanism in which a ketimine species is the common intermediate of half-transamination and β-elimination ofl-cysteine is proposed.l-cysteine partitions between these two reactions with a ratio of ~2.5. Rapid scanning stopped-flow and quench flow experiments permit the identification of reaction intermediates and the measurements of the kinetic parameters ofl-cysteine half-transamination. The k_cat of this reaction is 200- or 60-fold lower than that ofl-alanine andl-serine, respectively. Conversely,l-cysteine binds to AGT with a binding affinity 30- and 200-fold higher than that ofl-alanine andl-serine, respectively. This appears to be consistent with the calculated interaction energies of thel-cysteine,l-alanine andl-serine docked at the active site of AGT.

Keywords: Abbreviations; PLP; pyridoxal 5′-phosphate; AGT; alanine:glyoxylate aminotransferase; PMP; pyridoxamine 5′-phosphateAlanine:glyoxylate aminotransferase; Pyridoxal 5′-phosphate; α,β-Elimination; L; -cysteine

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BBA - Proteins and Proteomics (v.1784, #9)