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BBA - Proteins and Proteomics (v.1764, #8)

Editorial Board (pp. ii).

Reliable and controllable antibody fragment selections from Camelid non-immune libraries for target validation by Peter Verheesen; Andreas Roussis; Hans J. de Haard; Arjan J. Groot; Jord C. Stam; Johan T. den Dunnen; Rune R. Frants; Arie J. Verkleij; C. Theo Verrips; Silvère M. van der Maarel (pp. 1307-1319).

With the completion of the sequence of the human genome, emphasis is now switching to the human proteome. However, the number of proteins is not only larger than mRNAs in the transcriptome, proteins need often to be in complex with other proteins to be functional. A favourable option to study proteins in their natural context is with a combination of biochemical and microscopic techniques using specific antibodies. Therefore, we designed a fast, reliable and controllable selection and screening of single-domain antibody fragments (VHH) from a Camelid non-immune library. We isolated VHH for four muscle disease related proteins; emerin, actin, tropomyosin-1, and nuclear poly(A)-binding protein. Important features of antibodies for target validation studies are recognition of the antigen in natural conformations and biologically relevant complexes. We show that selected antibody fragments are functional in various immunological techniques and prove useful in diagnostic applications. Our selection strategy is amenable to automation and to the establishment of proteomics platforms. It opens the way to quickly and cost-effectively obtain multiple antibody fragments for many antigens that can detect changes in their localization, level, and modification as well as subtle changes in supramolecular structures, which often associate with disease.

Keywords: Llama heavy-chain antibodies; Non-immune library; Phage display; Polyclonal phage antibodies; Selection monitoring

A guest molecule–host cavity fitting algorithm to mine PDB for small molecule targets by William C. Byrem; Stephen C. Armstead; Shunji Kobayashi; Roderic G. Eckenhoff; David M. Eckmann (pp. 1320-1324).

Inhaled anesthetic molecule occupancy of a protein internal cavity depends in part on the volumes of the guest molecule and the host site. Current algorithms to determine volume and surface area of cavities in proteins whose structures have been determined and cataloged make no allowance for shape or small degrees of shape adjustment to accommodate a guest. We developed an algorithm to determine spheroid dimensions matching cavity volume and surface area and applied it to screen the cavities of 6,658 nonredundant structures stored in the Protein Data Bank (PDB) for potential targets of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane). Our algorithm determined sizes of prolate and oblate spheroids matching dimensions of each cavity found. If those spheroids could accommodate halothane (radius 2.91 Å) as a guest, we determined the packing coefficient. 394,766 total cavities were identified. Of 58,681 cavities satisfying the fit criteria for halothane, 11,902 cavities had packing coefficients in the range of 0.46–0.64. This represents 20.3% of cavities large enough to hold halothane, 3.0% of all cavities processed, and found in 2,432 protein structures. Our algorithm incorporates shape dependence to screen guest–host relationships for potential small molecule occupancy of protein cavities. Proteins with large numbers of such cavities are more likely to be functionally altered by halothane.

Keywords: Cavity; Host cavity; Protein cavity; Spheroid; Prolate; Oblate; Guest; Guest molecule; Algorithm; Halothane; CASTp

Chlorinations catalyzed by chloroperoxidase occur via diffusible intermediate(s) and the reaction components play multiple roles in the overall process by Kelath Murali Manoj (pp. 1325-1339).

The chlorination mechanism of the fungal enzyme chloroperoxidase (CPO) has been debated for (1) active site chlorination and (2) diffusible species mediated chlorination. Based upon the conversion of ∼35 different substrates belonging to different reactive groups, it was found that substrate dimensions and topography had no pronounced effect on rates of CPO chlorination reaction. Epoxidation of indene was dependent on its concentration where as chlorination was not. Also, effective conversion was seen in the chlorination mixture for substrates that could not be epoxidized or sulfoxidized. Some insoluble substrates and certain molecules that exceeded the active site dimensions were chlorinated at rates comparable to the rates required for CPO's more natural substrate, monochlorodimedone. By terminating the enzymatic reaction with an active site ligand (azide), the amount of diffusible species was correlated to CPO in the reaction mixture. The preferential utilization of a substrate, earlier attributed to the active site, is found to be due to the specificity afforded by the reaction environment. It was found that the reaction medium components of peroxide, chloride and hydronium ions affected the reaction rates through varying roles in the enzymatic and non-enzymatic process. Besides these experimental evidences, key mechanistic and kinetic arguments are presented to infer that the final chlorine transfer occurs outside the active site via a diffusible species.

Keywords: Chloroperoxidase; Haloperoxidase; Peroxidase; Chlorination mechanism and kinetics

Characterization of a fibrinolytic enzyme (ussurenase) from Agkistrodon blomhoffii ussurensis snake venom: Insights into the effects of Ca²⁺ on function and structure by Ming-Zhong Sun; Shuqing Liu; Frederick T. Greenaway (pp. 1340-1348).

Fibrino(geno)lytic enzymes from snake venoms have been identified as high quality therapeutic agents for treatment of blood clots and strokes. They act on fibrinogen and fibrin, leading to defibrinogenation of blood, lysis of fibrin, and a consequent decrease in blood viscosity. In this work, a fibrinolytic enzyme (ussurenase) from China Agkistrodon blomhoffii Ussurensis snake venom, was purified to homogeneity, identified as a stable 23,367.8 Da monomeric protein, and was identified as a new kind of snake venom metalloproteinase. Ussurenase reacts optimally with fibrin clots at pH 7.5–8.3 and a temperature of 33–41 °C. Although many fibrinolytic enzymes are known to be zinc-dependent, measurements from inductively coupled plasma-atomic emission spectroscopy (ICP-AES) reveal that ussurenase is a Ca²⁺-containing protein with a molar ratio of 1:1 ([Ca²⁺]:[enzyme]). Ca²⁺ is crucial to the fibrin clot hydrolysis by ussurenase but also plays an important role in maintaining the structural integrity of the enzyme. The addition of Ca²⁺ to the apoenzyme induces a conformational change making the environments surrounding the Trp residues of the enzyme more hydrophobic. The presence of Ca²⁺ also increases the structural stability of ussurenase, so that higher concentrations of the denaturant guanidine hydrochloride are required to denature the native ussurenase compared to the apo-form. UV absorption and CD spectroscopy experiments show that Ca²⁺ increases the thermostability and changes the secondary structure of ussurenase. All these data suggest that Ca²⁺ is crucial for the correct folding and activity of ussurenase.

Keywords: Abbreviations; A.b. Ussurensis; Agkistrodon blomhoffii Ussurensis; A. h. brevicaudus; Agkistrodon halys brevicaudus; A-50; DEAE Sephadex A-50; Bis; N,N; ′-methylenebisacrylamide; CD; Circular dichroism; EDTA; ethylenediaminetetraacetic acid; EGTA; ethylene glycol bis(2-aminoethyl ether)-; N,N; ,; N′N′; -tetraacetic acid; G-75; Sephadex G-75; ICP-AES; Inductively coupled plasma-atomic emission spectroscopy; MALDI-TOF MS; matrix-assisted laser desorption ionization time-of-flight mass spectrometry; NMR; nuclear magnetic resonance; PAGE; Polyacrylamide gel electrophoresis; PMF; protein mass fingerprint; S-300; Sephacryl 300; SDS-PAGE; Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Trp; tryptophan; Tyr; tyrosineUssurenase; Fibrinolytic enzyme; Snake venom; Characterization; Calcium ion; Function; Structure

Effect of disulfide-bond introduction on the activity and stability of the extended-spectrum class A β-lactamase Toho-1 by Akiko Shimizu-Ibuka; Hiroshi Matsuzawa; Hiroshi Sakai (pp. 1349-1355).

The production of class A β-lactamases is a major cause of clinical resistance to β-lactam antibiotics. Some of class A β-lactamases are known to have a disulfide bridge. Both narrow spectrum and extended spectrum β-lactamases of TEM and the SHV enzymes possess a disulfide bond between Cys77 and Cys123, and the enzymes with carbapenem-hydrolyzing activity have a well-conserved disulfide bridge between Cys69 and Cys238. We produced A77C/G123C mutant of the extended-spectrum β-lactamase Toho-1 in order to introduce a disulfide bond between the cysteine residues at positions 77 and 123. The result of 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) titrations confirmed formation of a new disulfide bridge in the mutant. The results of irreversible heat inactivation and circular dichroism (CD) melting experiments indicated that the disulfide bridge stabilized the enzyme significantly. Though kinetic analysis indicated that the catalytic properties of the mutant were quite similar to those of the wild-type enzyme, E. coli producing this mutant showed drug resistance significantly higher than E. coli producing the wild-type enzyme. We speculate that the stability of the enzymes provided by the disulfide bond may explain the wide distribution of TEM and SHV derivatives and explain how various mutations can cause broadened substrate specificity without loss of stability.

Keywords: Extended-spectrum β-lactamase; Disulfide bond; CD; Thermostability

The oxidised histone octamer does not form a H3 disulphide bond by Christopher M. Wood; Sirirath Sodngam; James M. Nicholson; Stanley J. Lambert; Colin D. Reynolds; John P. Baldwin (pp. 1356-1362).

A H3 dimer band is produced when purified native histone octamers are run on an SDS-PAGE gel in a β-mercaptoethanol-free environment. To investigate this, native histone octamer crystals, derived from chicken erythrocytes, and of structure (H2A–H2B)–(H4–H3)–(H3′–H4′)–(H2B′–H2A′), were grown in 2 M KCl, 1.35 M potassium phosphates and 250–350 μM of the oxidising agent S-nitrosoglutathione, pH 6.9. X-ray diffraction data were acquired to 2.10 Å resolution, yielding a structure with an R_work value of 18.6% and an R_free of 22.5%. The space group is P6₅, the asymmetric unit of which contains one complete octamer. Compared to the 1.90 Å resolution, unoxidised native histone octamer structure, the crystals show a reduction of 2.5% in the c-axis of the unit cell, and free-energy calculations reveal that the H3–H3′ dimer interface in the latter has become thermodynamically stable, in contrast to the former. Although the inter-sulphur distance of the two H3 cysteines in the oxidised native histone octamer has reduced to 6 Å from the 7 Å of the unoxidised form, analysis of the hydrogen bonds that constitute the (H4–H3)–(H3′–H4′) tetramer indicates that the formation of a disulphide bond in the H3–H3′ dimer interface is incompatible with stable tetramer formation. The biochemical and biophysical evidence, taken as a whole, is indicative of crystals that have a stable H3–H3′ dimer interface, possibly extending to the interface within an isolated H3–H3′ dimer, observed in SDS-PAGE gels.

Keywords: Abbreviations; CCLRC; Council for the Central Laboratory of the Research Councils; HO; histone octamer; GHO; GSNO-exposed histone octamer—the 2.10 Å histone octamer model (PDB code 2ARO) described herein; GSNO; S-nitrosoglutathione; NCP; nucleosome-core particle; OD; ₂₇₈; UV spectrographic measurement at 278 nm; PDB; protein database; RSH; any thiol; RSNO; any S-nitrosothiol; SM; starting model—the 1.90 Å histone octamer model (PDB code 1TZY)H3 dimer; Histone octamer; Hydrogen bond modulation; Oxidation; Reduction

Mass spectral analysis of the apolipoproteins on mouse high density lipoproteins. Detection of post-translational modifications by Donald L. Puppione; Lang M. Yam; Sara Bassilian; Puneet Souda; Lawrence W. Castellani; Verne N. Schumaker; Julian P. Whitelegge (pp. 1363-1371).

Using mass spectrometry, we have recently reported on molecular masses of the apolipoproteins associated with porcine and equine HDL. In addition to obtaining accurate masses for the various apolipoproteins, we also were able to detect mass variations due to post-translational modifications. In the present study, we have used these same approaches to characterize the apolipoproteins in two inbred mouse strains, C57BL/6 and BALB/c. Comparing our molecular mass data with calculated values for molecular weight, we were able to identify the correct sequences for several of the major apolipoproteins. Analyses were carried out on the apolipoproteins of ultracentrifugally isolated HDL. Prior to analyses by electrospray ionization mass spectrometry (ESI-MS), the apolipoproteins were separated either by size exclusion or reverse phase chromatography. The molecular masses of apoA-I, proapoA-I, apoA-II, proapoA-II, apoC-I and apoC-III were obtained. Comparing the values obtained for the two strains, differences in the molecular masses of apoA-I, apoA-II and apoC-III were observed. In this study, post-translationally modified apolipoproteins, involving loss of amino acids from both the N- and C-termini, oxidation of methionine residues and possible acylation, were noted following reverse-phase separation. Further analyses by tandem mass spectrometry (MSMS) done on the tryptic digests of apolipoproteins separated by reverse phase chromatography enabled us to confirm sequence differences between the two strains, to verify selected apoA-I sequences that had been entered into the GenBank and to identify which methionines in apoA-I, apoC-III and apoE had been converted to methionine sulfoxides.

Keywords: Propeptide; Proapolipoprotein; Methionine sulfoxide; Acylated apoA-I

Prediction of the mechanism of action of omuralide ( clasto-lactacystin β-lactone) on human cathepsin A based on a structural model of the yeast proteasome β5/PRE2-subunit/omuralide complex by Sei-ichi Aikawa; Fumiko Matsuzawa; Yurie Satoh; Yoshito Kadota; Hirofumi Doi; Kohji Itoh (pp. 1372-1380).

Cathepsin A (CathA) is a lysosomal serine carboxypeptidase that exhibits homology and structural similarity to the yeast and wheat serine carboxypeptidases (CPY and CPW) belonging to the α/β-hydrolase fold family. Human CathA (hCathA) and CPW have been demonstrated to be inhibited by a proteasome (threonine protease) inhibitor, lactacystin, and its active derivative, omuralide ( clasto-lactacystin β-lactone), as well as chymostatin. A hCathA/omuralide complex model constructed on the basis of the X-ray crystal structures of the CPW/chymostatin complex and the yeast proteasome β-subunit (β5/PRE2)/omuralide one predicted that the conformation of omuralide in the active-site cleft of proteasome β5/PRE2 should be very similar to that of chymostatin at the S1 catalytic subsites in the hCathA- and CPW-complexes. The relative positions of the glycine residues, i.e., Gly57 in hCathA, Gly53 in CPW, and Gly47 in β5/PRE2, present in the oxyanion hole of each enzyme were also highly conserved. These results suggest that omuralide might inhibit hCathA and CPW at the S1 subsite in their active-site clefts through direct binding to the active serine residue.

Keywords: Cathepsin A; Proteasome; Chymostatin; Lactacystin; Omuralide; Homology modeling

Identification of new inhibitors of E. coli cyclopropane fatty acid synthase using a colorimetric assay by Dominique Guianvarc'h; Thierry Drujon; Thearina Ear Leang; Fabienne Courtois; Olivier Ploux (pp. 1381-1388).

Bacterial cyclopropane synthases catalyze the cyclopropanation of unsaturated fatty acids by transferring a methylene group from S-adenosyl-l-methionine (AdoMet) to the double bond of the lipids. Mycobacterium tuberculosis cyclopropane synthases have been shown to be implicated in pathogenicity, and therefore constitute attractive targets for the development of new drugs against tuberculosis. However, no in vitro assay for these cyclopropane synthases has yet been described. The homologous E. coli enzyme, cyclopropane fatty acid synthase, is thus a valuable model for inhibitor screening. Here, we report the adaptation to the E. coli CFAS of a previously reported enzyme-coupled colorimetric assay based on the quantification, using Ellman's reagent, of homocysteine produced from S-adenosyl-l-homocysteine, a product of the reaction, in the presence of AdoHcy nucleosidase and S-ribosylhomocysteinase. Using this assay we measured the kinetic parameters for CFAS: K_m (AdoMet)=80 μM, k_cat=4 min−¹. We adapted this assay to microtiter plates and tested 15 potential inhibitors of CFAS. Among them, two new inhibitors, a lipid analog and a thioether analog of AdoHcy, showed IC₅₀ of 4 μM and 11 μM, respectively. This new assay will thus be useful for high-throughput screening of compound libraries for discovering novel antituberculous drug candidates.

Keywords: Abbreviations; AdoHcy; S; -adenosyl-; l; -homocysteine; AdoMet; S; -adenosyl-; l; -methionine; BSA; bovin serum albumin; CFAS; cyclopropane fatty acid synthase; CMAS; cyclopropane mycolic acid synthases; DMF; N; ,; N; -dimethylformamide; DMSO; dimethylsulfoxyde; DTNB; 5,5′-dithiobis-2-nitrobenzoic acid; DTT; dithiothreitol; EDTA; ethylenediaminetetraacetic acid; HPLC; high pressure liquid chromatography; TFA; trifluoroacetic acid; TNB; 2-nitro-5-thiobenzoic acidCyclopropane synthase; Inhibitor screening; 5,5′-dithiobis-2-nitrobenzoic acid; Spectrophotometric coupled assay; Homocysteine

Structural changes of α-lactalbumin induced by low pH and oleic acid by Fang Yang Jr.; Min Zhang; Jie Chen; Yi Liang (pp. 1389-1396).

The effects of low pH and oleic acid on conformation and association state of Ca²⁺-depleted bovine α-lactalbumin (apo-BLA) have been studied by electrospray ionization mass spectrometry, fluorescence spectroscopy, and circular dichroism. The experimental results demonstrate that two structurally distinct species exist in the conformational transition of apo-BLA induced by low pH. One species populates at pH 3.0 characterized as a monomeric molten globule state and the other accumulates at pH 4.0–4.5 which is a partially folded dimer. Oleic acid promotes the formation of the dimeric intermediate at pH 4.0 and 7.0, but increases the content of molten globule state remarkably at pH 3.0 compared with that in the absence of oleic acid, indicating that oleic acid at pH 3.0 plays a different role from those at pH 4.0 and 7.0. Our data provide insight into the mechanism of pH-dependent and oleic acid-dependent structural changes and oligomerization of α-lactalbumin, and will be helpful to the understanding of the apoptosis-inducing function of multimeric α-lactalbumin in which oleic acid is a necessary cofactor.

Keywords: Abbreviations; ANS; 8-anilino-1-naphthalene-sulfonic acid; apo-BLA; Ca; ²⁺; -depleted bovine α-lactalbumin; α-LA; α-Lactalbumin; ESI-TOF MS; electrospray ionization time-of-flight mass spectrometry; MALDI-TOF; matrix assisted laser desorption/ionization time-of-flight; CD; circular dichroism; HAMLET/BAMLET; h; uman/; b; ovine α-lactalbumin; m; ade; le; thal to; t; umor cells; MG; molten globuleα-Lactalbumin; Oleic acid; Electrospray ionization mass spectrometry; Protein unfolding; Fluorescence spectroscopy; Circular dichroism

Flexibility and enzymatic cold-adaptation: A comparative molecular dynamics investigation of the elastase family by Elena Papaleo; Laura Riccardi; Chiara Villa; Piercarlo Fantucci; Luca De Gioia (pp. 1397-1406).

Molecular dynamics simulations of representative mesophilic and psycrophilic elastases have been carried out at different temperatures to explore the molecular basis of cold adaptation inside a specific enzymatic family. The molecular dynamics trajectories have been compared and analyzed in terms of secondary structure, molecular flexibility, intramolecular and protein–solvent interactions, unravelling molecular features relevant to rationalize the efficient catalytic activity of psychrophilic elastases at low temperature. The comparative molecular dynamics investigation reveals that modulation of the number of protein–solvent interactions is not the evolutionary strategy followed by the psycrophilic elastase to enhance catalytic activity at low temperature. In addition, flexibility and solvent accessibility of the residues forming the catalytic triad and the specificity pocket are comparable in the cold- and warm-adapted enzymes. Instead, loop regions with different amino acid composition in the two enzymes, and clustered around the active site or the specificity pocket, are characterized by enhanced flexibility in the cold-adapted enzyme. Remarkably, the psycrophilic elastase is characterized by reduced flexibility, when compared to the mesophilic counterpart, in some scattered regions distant from the functional sites, in agreement with hypothesis suggesting that local rigidity in regions far from functional sites can be beneficial for the catalytic activity of psychrophilic enzymes.

Keywords: Molecular dynamics simulations; Serine protease; Elastase; Cold adaptation; Psychrophilic enzyme; Flexibility

The secondary structure of pressure- and temperature-induced aggregates of equine serum albumin studied by FT-IR spectroscopy by Akira Okuno; Minoru Kato; Yoshihiro Taniguchi (pp. 1407-1412).

The protein aggregation is divided into amyloid fibrils and amorphous aggregates. Amyloid fibrils are composed of the 3-dimensional ordered structure and are bound to thioflavin T and Congo red dyes. The amorphous aggregates with the disordered structure do not bind to these dyes. We have investigated the pressure- and heat-induced aggregates of equine serum albumin (ESA) from the secondary structural viewpoint using FT-IR spectroscopy. We show the secondary structural differences between heat- and pressure-induced aggregates of ESA. The heat-induced irreversible aggregates of ESA are composed of the intermolecular β-sheet structure without binding thioflavie T and Congo red to be amorphous form. On the other hand, the pressure-induced reversible aggregates are composed of the random structure to be also amorphous form. From the comparison of pressure effects on ESA in native and reducing conditions of disulfide bridges, we demonstrate that the restriction of structural flexibility by disulfide bridges is an important factor for the reversibility of the pressure-induced aggregation.

Keywords: Serum albumin; High pressure FT-IR; Pressure-induced protein aggregation; Heat-induced protein aggregation; Structural flexibility

Expression, purification, kinetic, and structural characterization of an α-class carbonic anhydrase from Aedes aegypti (AaCA1) by S. Zoë Fisher; Iyerus Tariku; Nicolette M. Case; Chingkuang Tu; Teri Seron; David N. Silverman; Paul J. Linser; Robert McKenna (pp. 1413-1419).

Carbonic anhydrases (CAs) are zinc-containing metalloenzymes that catalyze the interconversion of carbon dioxide and bicarbonate. The α-class CAs are found predominantly in vertebrates, but they are also expressed in insects like mosquitoes. Recently, an α-CA from the midgut of Aedes aegypti larvae (AaCA1) was identified, cloned, and subsequently shown to share high sequence homologous to human CA I (HCA I). This paper presents the bacterial expression, purification, and kinetic characterization of the soluble CA domain of AaCA1. The data show AaCA1 is a highly active CA that displays inhibition by methazolamide and ethoxzolamide with nM affinity. Additionally, a homology model of AaCA1, based on the crystal structure of HCA I, is presented and the overall structure, active site, and surface charge properties are compared to those of HCA I and II. Measurements of catalysis show that AaCA1 is more like HCA II in terms of proton transfer, but more similar to HCA I in terms of conversion of carbon dioxide to bicarbonate, and these differences are rationalized in terms of structure. These results also indicate that amino acid differences in the active site of AaCA1 compared to human CAs could be used to design specific CA inhibitors for the management of mosquito populations.

Keywords: Carbonic anhydrase; Mosquito; Gut alkalinization

The combined effect of fibrin formation and factor XIII A subunit Val34Leu polymorphism on the activation of factor XIII in whole plasma by Amir H. Shemirani; Gizella Haramura; Zsuzsa Bagoly; László Muszbek (pp. 1420-1423).

The first step in the activation of blood coagulation factor XIII (FXIII) is the proteolytic cleavage of the potentially active A subunit (FXIII-A) by thrombin at Arg37–Gly38. Both fibrin formation and FXIII-A Val34Leu polymorphism influence the rate of proteolytic activation of purified factor XIII, however their relative importance and interaction in determining the time of onset and the rate of FXIII activation in whole plasma have not yet been explored. In the present study it was shown that in plasma, fibrin formation preceded the truncation of FXIII-A by thrombin, the activation process took place exclusively on the surface of newly formed fibrin and activated FXIII remained associated with the fibrin clot. The time of fibrin formation closely correlated with the time of FXIII activation, while there was no significant relationship between the time of FXIII activation and FXIII-A Val34Leu genotype. However, in the case of Leu34 variant the lag phase between fibrin formation and FXIII-A truncation was significantly shorter than in the case of Val34 variant. The results suggest that in whole plasma the onset of FXIII activation is determined by fibrin formation, while the rate of activation is modulated by Val34Leu polymorphism.

Keywords: Factor XIII Val34Leu polymorphism; Fibrinogen; Fibrin polymerization; Thrombin; Transglutaminase

Implication of substrate-assisted catalysis on improving lipase activity or enantioselectivity in organic solvents by Shau-Wei Tsai; Chun-Chi Chen; Hung-Shien Yang; I-Son Ng; Teh-Liang Chen (pp. 1424-1428).

In comparison with the biocatalyst engineering and medium engineering approaches, very few examples have been reported on using the substrate engineering approach such as substrate-assisted catalysis (SAC) for naturally occurring or engineered lipases and serine proteases to improve the enzyme activity and enantioselectivity. By employing lipase-catalyzed hydrolysis of ( R, S)-naproxen esters in water-saturated isooctane as the model system, we demonstrate the proton shuttle device to the leaving alcohol of the substrate as a new means of SAC to effectively improve the lipase activity or enantioselectivity. The result cannot only provide a strong evidence for the rate-limiting proton transfer for the bond-breaking of tetrahedron intermediate of the acylation step, but also sheds light for performing the hydrolysis, transesterification or aminolysis in organic solvents for the ester substrate that originally lipases cannot catalyze, but now can after introducing the device.

Keywords: Abbreviations; SAC; substrate-assisted catalysis; pCPL; Carica papaya; lipase; pCPHL; Carica pentagona; Heilborn lipaseLipases; Substrate-assisted catalysis; Proton transfer; Acylation step; Hydrolysis resolution

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BBA - Proteins and Proteomics (v.1764, #8)