The lid is a structural and functional determinant of lipase activity and selectivity

In several lipases access to the enzyme active site is regulated by the position of a mobile structure named the lid. The role of this region in modulating lipase function is reviewed in this paper analysing the results obtained with three different recombinant lipases modified in the lid sequence: Candida rugosa lipase isoform 1 (CRL1), Pseudomonas fragi lipase (PFL) and Bacillus subtilis lipase A (BSLA). A CRL chimera enzyme obtained by replacing its lid with that of another C. rugosa lipase isoform (CRL1LID3) was found to be affected in both activity and enantioselectivity in organic solvent. Variants of the PFL protein in which three polar lid residues were replaced with amino acids strictly conserved in homologous lipases displayed altered chain length preference profile and increased thermostability. On the other hand, insertion of lid structures from structurally homologous enzymes into BSLA, a lipase that naturally does not possess such a lid structure, caused a reduction in the enzyme activity and an altered substrate specificity. These results strongly support the concept that the lid plays an important role in modulating not only activity but also specifity, enantioselectivity and stability of lipase enzymes.     

Inhibition of yeast lipase (CRL1) and cholesterol esterase (CRL3) by 6-chloro-2-pyrones: comparison with porcine cholesterol esterase

Previously, it was demonstrated that pancreatic cholesterol esterase is selectively inhibited by 6-chloro-2-pyrones with cyclic aliphatic substituents in the 3-position. Inhibition is reversible and is competitive with substrate. Pancreatic cholesterol esterase is a potential target for treatment of hypercholesterolemia. In the present study, yeast cholesterol esterase from Candida cylindracea (also called C. rugosa CRL3) was compared to porcine pancreatic cholesterol esterase for inhibition by a series of 3-alkyl- or 5-alkyl-6-chloro-2-pyrones. In addition, CRL3 was compared with the related yeast lipase CRL1. Inhibition of CRL3 by substituted 6-chloro-2-pyrones was competitive with binding of the substrate p-nitrophenyl butyrate. Inhibition constants ranged from 0.2 microM to >90 microM. Small changes in the alkyl group had profound effects on binding. The pattern of inhibition of CRL3 is quite distinct from that observed with porcine cholesterol esterase. Molecular modeling studies suggest that the orientation of binding of these inhibitors at the active site of CRL3 can vary but that the pyrone ring consistently occupies a position close to the active site serine. CRL1 is highly homologous to CRL3. Nevertheless, patterns of inhibition of CRL1 by substituted 6-chloro-2-pyrones differ markedly from patterns observed with CRL3. The substituted 6-chloro-2-pyrones are slowly hydrolyzed in the presence of CRL1 and are pseudosubstrates of CRL3, but are simple reversible inhibitors of pancreatic cholesterol esterase     

A critical role for the histidine residues in the catalytic function of acyl-CoA:cholesterol acyltransferase catalysis: evidence for catalytic difference between ACAT1 and ACAT2

To investigate a role for histidine residues in the expression of normal acyl-CoA:cholesterol acyltransferase (ACAT) activity, the histidine residues located at five different positions in two isoenzymes were substituted by alanine, based on the sequence homology between ACAT1 and ACAT2. Among the 10 mutants generated by baculovirus expression technology, H386A-ACAT1, H460A-ACAT1, H360A-ACAT2, and H399A-ACAT2 lost their enzymatic activity completely. A reduction in catalytic activity is unlikely to result from structural changes in the substrate-binding pocket, because their substrate-binding affinities were normal. However, the enzymatic activity of H386A-ACAT1 was restored to <37% of the level of the wild-type activity when cholesterol was replaced by 25-hydroxycholesterol as substrate. H527A-ACAT1 and H501A-ACAT2, termed carboxyl end mutants, exhibit activities of ∼96% and ∼75% of that of the wild-type. Interestingly, H425A-ACAT1 showed 59% of the wild-type activity, in contrast to its equivalent mutant, H399A-ACAT2. These results demonstrate that the histidine residues located at the active site are very crucial both for the catalytic activity of the enzyme and for distinguishing ACAT1 from ACAT2 with respect to enzyme catalysis and substrate specificity.     

The triglyceride lipases of the pancreas

Pancreatic triglyceride lipase (PTL) and its protein cofactor, colipase, are required for efficient dietary triglyceride digestion. In addition to PTL, pancreatic acinar cells synthesize two pancreatic lipase related proteins (PLRP1 and PLRP2), which have a high degree of sequence and structural homology with PTL. PLRP1 has no known activity. PTL and PLRP2 differ in substrate specificity, behavior in bile salts and dependence on colipase. Each protein has a globular amino-terminal (N-terminal) domain, which contains the catalytic site for PTL and PLRP2, and a beta-sandwich carboxyl-terminal (C-terminal) domain, which includes the predominant colipase-binding site for PTL. Inactive and active conformations of PTL have been described. They differ in the position of a surface loop, the lid domain, and of the beta5-loop. In the inactive conformation, the lid covers the active site and, upon activation by bile salt micelles and colipase or by lipid-water interfaces, the lid moves dramatically to open and configure the active site. After the lid movement, PTL and colipase create a large hydrophobic plateau that can interact with the lipid-water interface. A hydrophobic surface loop in the C-terminal domain, the beta5' loop, may also contribute to the interfacial-binding domain of the PTL-colipase complex.     

Evolution of Stability in a Cold-Active Enzyme Elicits Specificity Relaxation and Highlights Substrate-Related Effects on Temperature Adaptation

Molecular aspects of thermal adaptation of proteins were studied by following the co-evolution of temperature dependence, conformational stability, and substrate specificity in a cold-active lipase modified via directed evolution. We found that the evolution of kinetic stability was accompanied by a relaxation in substrate specificity. Moreover, temperature dependence and selectivity turned out to be mutually dependent. While the wild-type protein was strictly specific for short-chain triglycerides (C4) in the temperature range 10-50 °C and displayed highest activity in the cold, its stabilized variant was able to accept C8 and C12 molecules and its selectivity was temperature dependent. We could not detect any improvement in the overall structural robustness of the mutant when the structure was challenged by temperature or chemical denaturants. There is, however, strong evidence for local stabilization effects in the active-site region provided by two independent approaches. Differential scanning fluorimetry revealed that the exposure of hydrophobic patches (as the active site is) precedes denaturation, and molecular dynamics simulations confirmed that stability was obtained by restriction of the mobility of the lid, a flexible structure that regulates the access to the enzyme active site and influences its stability. This reduction of lid movements is suggested to be accompanied by a concomitant increase in the mobility of other protein regions, thus accounting for the observed broadening of substrate specificity.     

Substrate specificity of lipoprotein lipase and endothelial lipase: studies of lid chimeras

The triglyceride (TG) lipase gene subfamily, consisting of LPL, HL, and endothelial lipase (EL), plays a central role in plasma lipoprotein metabolism. Compared with LPL and HL, EL is relatively more active as a phospholipase than as a TG lipase. The amino acid loop or "lid" covering the catalytic site has been implicated as the basis for the difference in substrate specificity between HL and LPL. To determine the role of the lid in the substrate specificity of EL, we studied EL in comparison with LPL by mutating specific residues of the EL lid and exchanging their lids. Mutation studies showed that amphipathic properties of the lid contribute to substrate specificity. Exchanging lids between LPL and EL only partially shifted the substrate specificity of the enzymes. Studies of a double chimera possessing both the lid and the C-terminal domain (C-domain) of EL in the LPL backbone showed that the role of the lid in determining substrate specificity does not depend on the nature of the C-domain of the lipase. Using a kinetic assay, we showed an additive effect of the EL lid on the apparent affinity for HDL(3) in the presence of the EL C-domain.     

Oxalate decarboxylase and oxalate oxidase activities can be interchanged with a specificity switch of up to 282,000 by mutating an active site lid

Oxalate decarboxylases and oxalate oxidases are members of the cupin superfamily of proteins that have many common features: a manganese ion with a common ligand set, the substrate oxalate, and dioxygen (as either a unique cofactor or a substrate). We have hypothesized that these enzymes share common catalytic steps that diverge when a carboxylate radical intermediate becomes protonated. The Bacillus subtilis decarboxylase has two manganese binding sites, and we proposed that Glu162 on a flexible lid is the site 1 general acid. We now demonstrate that a decarboxylase can be converted into an oxidase by mutating amino acids of the lid that include Glu162 with specificity switches of 282,000 (SEN161-3DAS), 275,000 (SENS161-4DSSN), and 225,000 (SENS161-4DASN). The structure of the SENS161-4DSSN mutant showed that site 2 was not affected. The requirement for substitutions other than of Glu162 was, at least in part, due to the need to decrease the Km for dioxygen for the oxidase reaction. Reversion of decarboxylase activity could be achieved by reintroducing Glu162 to the SENS161-4DASN mutant to give a relative specificity switch of 25,600. This provides compelling evidence for the crucial role of Glu162 in the decarboxylase reaction consistent with it being the general acid, for the role of the lid in controlling the Km for dioxygen, and for site 1 being the sole catalytically active site. We also report the trapping of carboxylate radicals produced during turnover of the mutant with the highest oxidase activity. Such radicals were also observed with the wild-type decarboxylase.     

Design and realization of a tailor-made enzyme to modify the molecular recognition of 2-arylpropionic esters by Candida rugosa lipase

Within a research project aimed at probing the substrate specificity and the enantioselectivity of Candida rugosa lipase (CRL), computer modeling studies of the interactions between CRL and methyl (+/-)-2-(3-benzoylphenyl)propionate (Ketoprofen methyl ester) have been carried out in order to identify which amino acids are essential to the enzyme/substrate interaction. Different binding models of the substrate enantiomers to the active site of CRL were investigated by applying a computational protocol based on molecular docking, conformational analysis, and energy minimization procedures. The structural models of the computer generated complexes between CRL and the substrates enabled us to propose that Phe344 and Phe345, in addition to the residues constituting the catalytic triad and the oxyanion hole, are the amino acids mainly involved in the enzyme-ligand interactions. To test the importance of these residues for the enzymatic activity, site-directed mutagenesis of the selected amino acids has been performed, and the mutated enzymes have been evaluated for their conversion and selectivity capabilities toward different substrates. The experimental results obtained in these biotransformation reactions indicate that Phe344 and especially Phe345 influence CRL activity, supporting the findings of our theoretical simulations.     

Reactivity of pure Candida rugosa lipase isoenzymes (Lip1, Lip2, and Lip3) in aqueous and organic media. influence of the isoenzymatic profile on the lipase performance in organic media

Three pure isoenzymes from Candida rugosa lipase (CRL: Lip1, Lip2, and Lip3) were compared in terms of their stability and reactivity in both aqueous and organic media. The combined effect of temperature and pH on their stability was studied applying a factorial design. The analysis of the response surfaces indicated that Lip1 and Lip3 have a similar stability, lower than that of Lip2. In aqueous media, Lip3 was the most active enzyme on the hydrolysis of p-nitrophenyl esters, whereas Lip1 showed the highest activity on the hydrolysis of most assayed triacylglycerides. The highest differences among isoenzymes were found in the hydrolysis of triacylglycerides. Thus, a short, medium, and long acyl chain triacylglyceride was the preferred substrate for Lip3, Lip1, and Lip2, respectively. In organic medium, Lip3 and Lip1 provided excellent results in terms of enantioselectivity in the resolution of ibuprofen (EF value over 0.90) and conversion, whereas initial esterification rate was higher for Lip3. However, the use of Lip2 resulted in lower values of conversion, enantiomeric excess, and enantioselectivity. In the case of trans-2-phenyl-1-cyclohexanol (TPCH) resolution, initial esterification rates were high except for Lip3, which also produced poor results in conversion and enantiomeric excess. The performance of the pure isoenzymes in the enantioselectivity esterification of these substrates was compared with different CRL crude preparations with known isoenzymatic content and the different results could not be explained by their isoenzymatic profile. Therefore, it can be concluded that other factors can also affect the catalysis of CRL and only the reproducibility between powders can ensure the reproducibility in synthesis reactions.     

Crystal structure of a mono- and diacylglycerol lipase from Malassezia globosa reveals a novel lid conformation and insights into the substrate specificity

Most lipases contain a lid domain to shield the hydrophobic binding site from the water environment. The lid, mostly in helical form, can undergo a conformational change to expose the active cleft during the interfacial activation. Here we report the crystal structures of Malassezia globosa LIP1 (SMG1) at 1.45 and 2.60 ? resolution in two crystal forms. The structures present SMG1 in its closed form, with a novel lid in loop conformation. SMG1 is one of the few members in the fungal lipase family that has been found to be strictly specific for mono- and diacylglycerol. To date, the mechanism for this substrate specificity remains largely unknown. To investigate the substrate binding properties, we built a model of SMG1 in open conformation. Based on this model, we found that the two bulky hydrophobic residues adjacent to the catalytic site and the N-terminal hinge region of the lid both may act as steric hindrances for triacylglycerols binding. These unique structural features of SMG1 will provide a better understanding on the substrate specificity of mono- and diacylglycerol lipases and a platform for further functional study of this enzyme.     

Activity of 3-ketosteroid 9α-hydroxylase (KshAB) indicates cholesterol side chain and ring degradation occur simultaneously in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb), a significant global pathogen, contains a cholesterol catabolic pathway. Although the precise role of cholesterol catabolism in Mtb remains unclear, the Rieske monooxygenase in this pathway, 3-ketosteroid 9α-hydroxylase (KshAB), has been identified as a virulence factor. To investigate the physiological substrate of KshAB, a rhodococcal acyl-CoA synthetase was used to produce the coenzyme A thioesters of two cholesterol derivatives: 3-oxo-23,24-bisnorchol-4-en-22-oic acid (forming 4-BNC-CoA) and 3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid (forming 1,4-BNC-CoA). The apparent specificity constant (k(cat)/K(m)) of KshAB for the CoA thioester substrates was 20-30 times that for the corresponding 17-keto compounds previously proposed as physiological substrates. The apparent K(m)(O(2)) was 90 ± 10 μM in the presence of 1,4-BNC-CoA, consistent with the value for two other cholesterol catabolic oxygenases. The Δ(1) ketosteroid dehydrogenase KstD acted with KshAB to cleave steroid ring B with a specific activity eight times greater for a CoA thioester than the corresponding ketone. Finally, modeling 1,4-BNC-CoA into the KshA crystal structure suggested that the CoA moiety binds in a pocket at the mouth of the active site channel and could contribute to substrate specificity. These results indicate that the physiological substrates of KshAB are CoA thioester intermediates of cholesterol side chain degradation and that side chain and ring degradation occur concurrently in Mtb. This finding has implications for steroid metabolites potentially released by the pathogen during infection and for the design of inhibitors for cholesterol-degrading enzymes. The methodologies and rhodococcal enzymes used to generate thioesters will facilitate the further study of cholesterol catabolism.     

Stereospecific oxidation of secondary alcohols by human alcohol dehydrogenases

The human liver alpha alpha alcohol dehydrogenase exhibits a different substrate specificity and stereospecificity for secondary alcohols than the human beta 1 beta 1, and gamma 1 gamma 1 or horse liver alcohol dehydrogenases. All of the enzymes efficiently oxidize primary alcohols, but alpha alpha oxidizes secondary alcohols far more efficiently than human beta 1 beta 1 and gamma 1 gamma 1 or horse liver alcohol dehydrogenase. Specifically, alpha alpha oxidizes four- and five-carbon secondary alcohols with efficiencies 0.06-2.2 times that of primary homologs and oxidizes these secondary alcohols with efficiencies up to 3 orders of magnitude greater than those of the three other isoenzymes. Whereas the human beta 1 beta 1, gamma 1 gamma 1 and horse isoenzymes show a distinct preference toward (S)-(+)-3-methyl-2-butanol, the alpha alpha isoenzyme prefers (R)-(-)-3-methyl-2-butanol. Computer-simulated graphics demonstrate that the horse subunit accommodates (S)-(+)-3-methyl-2-butanol within the active site much better than the opposite stereoisomer, primarily due to steric hindrance caused by Phe-93. Human alpha may accommodate (R)-(-)-3-methyl-2-butanol better than (S)-(+)-3-methyl-2-butanol because of close contacts between the latter and Thr-48. These observations suggest that substitutions at positions 93 and 48 in the active site of human liver alcohol dehydrogenase isoenzymes may determine their substrate specificity for secondary alcohols.     

Yeast D-amino acid oxidase: structural basis of its catalytic properties

The 3D structure of the flavoprotein D-amino acid oxidase (DAAO) from the yeast Rhodotorula gracilis (RgDAAO) in complex with the competitive inhibitor anthranilate was solved (resolution 1.9A) and structural features relevant for the overall conformation and for catalytic activity are described. The FAD is bound in an elongated conformation in the core of the enzyme. Two anthranilate molecules are found within the active site cavity; one is located in a funnel forming the entrance, and the second is in contact with the flavin. The anchoring of the ligand carboxylate with Arg285 and Tyr223 is found for all complexes studied. However, while the active site group Tyr238-OH interacts with the carboxylate in the case of the substrate D-alanine, of D-CF(3)-alanine, or of L-lactate, in the anthranilate complex the phenol group rotates around the C2-C3 bond thus opening the entrance of the active site, and interacts there with the second bound anthranilate. This movement serves in channeling substrate to the bottom of the active site, the locus of chemical catalysis. The absence in RgDAAO of the "lid" covering the active site, as found in mammalian DAAO, is interpreted as being at the origin of the differences in kinetic mechanism between the two enzymes. This lid has been proposed to regulate product dissociation in the latter, while the side-chain of Tyr238 might exert a similar role in RgDAAO. The more open active site architecture of RgDAAO is the origin of its much broader substrate specificity. The RgDAAO enzyme forms a homodimer with C2 symmetry that is different from that reported for mammalian D-amino acid oxidase. This different mode of aggregation probably causes the differences in stability and tightness of FAD cofactor binding between the DAAOs from different sources.     

Author index of volume 26, 2008

The stereospecific esterification of ibuprofen by Candida rugosa lipase (CRL) with 1-butanol was optimised. The yield of the butyl ester was nearly quantitative with an enantiomeric excess of 95% and E 100. Enzyme desiccation over P₂O₅ had a pronounced effect on the esterification yield and its role in stereospecificity is discussed. Molecular modelling and energy-minimisation studies were also performed to understand the stereospecific binding of substrates to the active site of CRL. The docking of the substrate S(+) ibuprofen to the active site of CRL was performed based on the three-dimensional structure of CRL (PDB entry 1CRL). The results show that only the active S(+) enantiomer of ibuprofen is able to form direct contacts with the reactive hydroxyl group (Ser209) and imidazole base (His449) at the active site, whereas with the R enantiomer the functional group COOH points away from the (His449) base of CRL. This is in accordance with experimental results which show that the stereospecifity of CRL is towards S(-) ibuprofen.     

Extended interactions with prothrombinase enforce affinity and specificity for its macromolecular substrate

The specific action of serine proteinases on protein substrates is a hallmark of blood coagulation and numerous other physiological processes. Enzymic recognition of substrate sequences preceding the scissile bond is considered to contribute dominantly to specificity and function. We have investigated the contribution of active site docking by unique substrate residues preceding the scissile bond to the function of prothrombinase. Mutagenesis of the authentic P(1)-P(3) sequence in prethrombin 2/fragment 1.2 yielded substrate variants that could be converted to thrombin by prothrombinase. Proteolytic activation was also observed with a substrate variant containing the P(1)-P(3) sequence found in a coagulation zymogen not known to be activated by prothrombinase. Lower rates of activation of the variants derived from a decrease in maximum catalytic rate but not in substrate affinity. Replacement of the P(1) residue with Gln yielded an uncleavable derivative that retained the affinity of the wild type substrate for prothrombinase but did not engage the active site of the enzyme. Thus, active site docking of the substrate contributes to catalytic efficiency, but it is does not determine substrate affinity nor does it fully explain the specificity of prothrombinase. Therefore, extended interactions between prothrombinase and substrate regions removed from the cleavage site drive substrate affinity and enforce the substrate specificity of this enzyme complex.     

Structural basis for differences in substrate specificity of aspartate aminotransferase isoenzymes

X-Ray structural data concerning the substrate binding site of cytosolic chicken aspartate aminotransferase (AspAT) are reported. The structure of the complex of AspAT with the substrate-like inhibitor maleate has been refined at 2.2 A resolution. The lengths of hydrogen bonds between a bound molecule of maleate and side chains of amino acid residues in the active site are presented as well as other interatomic distances in the substrate binding site. The data obtained for the cytosolic AspAT have been compared with those for the mitochondrial chicken AspAT. It has been inferred that differences in substrate specificity of the AspAT isoenzymes are determined by interactions involving amino acid residues which are situated in the immediate vicinity of the active site and influence ionization or orientation of functional groups interacting with substrate. An explanation is suggested for different rates of transamination of aromatic amino acids in the active sites of the cytosolic and mitochondrial isoenzymes.     

Understanding Candida rugosa lipases: an overview

Candida rugosa lipase (CRL) is one of the enzymes most frequently used in biotransformations. However, there are some irreproducibility problems inherent to this biocatalyst, attributed either to differences in lipase loading and isoenzymatic profile or to other medium-engineering effects (temperature, a(w), choice of solvent, etc.). In addition, some other properties (influence of substrate and reaction conditions on the lid movement, differences in the glycosylation degree, post-translational modifications) should not be ruled out. In the present paper the recent developments published in the CRL field are overviewed, focusing on: (a) comparison of structural and biochemical data among isoenzymes (Lip1-Lip5), and their influence in the biocatalytical performance; (b) developments in fermentation technology to achieve new crude C. rugosa lipases; (c) biocatalytical reactivity of each isoenzyme, and methods for characterising them in crude CRL; (d) state-of-the-art of new applications performed with recombinant CRLs, both in CRL-second generation (wild-type recombinant enzymes), as well as in CRL-third generation, (mutants of the wt-CRL).     

Serine hydroxymethyltransferase from Escherichia coli: purification and properties.

Serine hydroxymethyltransferase from Escherichia coli was purified to homogeneity. The enzyme was a homodimer of identical subunits with a molecular weight of 95,000. The amino acid sequence of the amino and carboxy-terminal ends and the amino acid composition of cysteine-containing tryptic peptides were in agreement with the primary structure proposed for this enzyme from the structure of the glyA gene (M. Plamann, L. Stauffer, M. Urbanowski, and G. Stauffer, Nucleic Acids Res. 11:2065-2074, 1983). The enzyme contained no disulfide bonds but had one sulfhydryl group on the surface of the protein. Several sulfhydryl reagents reacted with this exposed group and inactivated the enzyme. Spectra of the enzyme in the presence of substrates and substrate analogs showed that the enzyme formed the same complexes and in similar relative concentrations as previously observed with the cytosolic and mitochondrial rabbit liver isoenzymes. Kinetic studies with substrates showed that the affinity and synergistic binding of the amino acid and folate substrates were similar to those obtained with the rabbit liver isoenzymes. The enzyme catalyzed the cleavage of threonine, allothreonine, and 3-phenylserine to glycine and the corresponding aldehyde in the absence of tetrahydrofolate. The enzyme was also inactivated by D-alanine caused by the transamination of the active site pyridoxal phosphate to pyridoxamine phosphate. This substrate specificity was also observed with the rabbit liver isoenzymes. We conclude that the reaction mechanism and the active site structure of E. coli serine hydroxymethyltransferase are very similar to the mechanism and structure of the rabbit liver isoenzymes.     

Identification of Crucial Amino Acids in Mouse Aldehyde Oxidase 3 That Determine Substrate Specificity

In order to elucidate factors that determine substrate specificity and activity of mammalian molybdo-flavoproteins we performed site directed mutagenesis of mouse aldehyde oxidase 3 (mAOX3). The sequence alignment of different aldehyde oxidase (AOX) isoforms identified variations in the active site of mAOX3 in comparison to other AOX proteins and xanthine oxidoreductases (XOR). Based on the structural alignment of mAOX3 and bovine XOR, differences in amino acid residues involved in substrate binding in XORs in comparison to AOXs were identified. We exchanged several residues in the active site to the ones found in other AOX homologues in mouse or to residues present in bovine XOR in order to examine their influence on substrate selectivity and catalytic activity. Additionally we analyzed the influence of the [2Fe-2S] domains of mAOX3 on its kinetic properties and cofactor saturation. We applied UV-VIS and EPR monitored redox-titrations to determine the redox potentials of wild type mAOX3 and mAOX3 variants containing the iron-sulfur centers of mAOX1. In addition, a combination of molecular docking and molecular dynamic simulations (MD) was used to investigate factors that modulate the substrate specificity and activity of wild type and AOX variants. The successful conversion of an AOX enzyme to an XOR enzyme was achieved exchanging eight residues in the active site of mAOX3. It was observed that the absence of the K889H exchange substantially decreased the activity of the enzyme towards all substrates analyzed, revealing that this residue has an important role in catalysis.     

Thiol-activated serine proteinases from nymphal hemolymph of the African migratory locust,Locusta migratoria migratorioides

Two unique serine proteinase isoenzymes (LmHP-1 and LmHP-2) were isolated from the hemolymph of African migratory locust (Locusta migratoria migratorioides) nymphs. Both have a molecular mass of about 23 kDa and are activated by thiol-reducing agents. PMSF abolishes enzymes activity only after thiol activation, while the cysteine proteinase inhibitors E-64, iodoacetamide, and heavy metals fail to inhibit the thiol-activated enzymes. The N-terminal sequence was determined for the more-abundant LmHP-2 isoenzyme. It exhibits partial homology to that of other insect serine proteinases and similar substrate specificity and inhibition by the synthetic and protein trypsin inhibitors pABA, TLCK, BBI, and STI. The locust trypsins LmHP-1 and LmHP-2 constitute a new category of serine proteases wherein the active site of the enzyme is exposed by thiol activation without cleavage of peptide bonds.