Topological and functional characterization of WbpM, an inner membrane UDP-GlcNAc C6 dehydratase essential for lipopolysaccharide biosynthesis in Pseudomonas aeruginosa

WbpM is essential for the biosynthesis of B-band lipopolysaccharide (LPS) in many serotypes of Pseudomonas aeruginosa. Homologues that can functionally complement a wbpM null mutant and that are also necessary for virulence have been identified in numerous pathogenic bacteria. WbpM and most of its homologues are large membrane proteins, which has long hampered the elucidation of their biochemical function. This paper describes the detailed characterization of WbpM using both in vivo and in vitro approaches. LacZ and PhoA fusion experiments showed that WbpM was anchored to the inner membrane via four N-terminal transmembrane domains, whereas the C-terminal catalytic domain resided in the cytoplasm. Although the membrane domains did not have any catalytic activity, complementation experiments suggested that they were important for the polymerization of high-molecular-weight B-band LPS. The biochemical characterization of a soluble truncated form of WbpM, His-S262, showed that WbpM was a C6 dehydratase specific for UDP-GlcNAc. It exhibited unusual low temperature (25-30 degrees C) and high pH (pH 10) optima. Although WbpM possessed an altered catalytic triad composed of SMK as opposed to SYK commonly found in other dehydratases, its catalysis was very efficient, with a kcat of 168 min(-1) and a kcat/Km of 58 mM(-1) min(-1). These unusual physico-kinetic properties suggested a potentially different mechanism of C6 dehydration for WbpM and its large homologues. His-S262 is now a precious tool for further structure-function studies.     

Completion of the canonical pathway for assembly of anticancer drugs vincristine/vinblastine in Catharanthus roseus

Glycosylation is a key modification for most molecules including plant natural products, for example, flavonoids and isoflavonoids, and can enhance the bioactivity and bioavailability of the natural products. The crystal structure of plant rhamnosyltransferase UGT89C1 from Arabidopsis thaliana was determined, and the structures of UGT89C1 in complexes with UDP‐β‐l ‐rhamnose and acceptor quercetin revealed the detailed interactions between the enzyme and its substrates. Structural and mutational analysis indicated that Asp356, His357, Pro147 and Ile148 are key residues for sugar donor recognition and specificity for UDP‐β‐l ‐rhamnose. The mutant H357Q exhibited activity with both UDP‐β‐l ‐rhamnose and UDP‐glucose. Structural comparison and mutagenesis confirmed that His21 is a key residue as the catalytic base and the only catalytic residue involved in catalysis independently as UGT89C1 lacks the other catalytic Asp that is highly conserved in other reported UGTs and forms a hydrogen bond with the catalytic base His. Ser124 is located in the corresponding position of the catalytic Asp in other UGTs and is not able to form a hydrogen bond with His21. Mutagenesis further showed that Ser124 may not be important in its catalysis, suggesting that His21 and acceptor may form an acceptor‐His dyad and UGT89C1 utilizes a catalytic dyad in catalysis instead of catalytic triad. The information of structure and mutagenesis provides structural insights into rhamnosyltransferase substrate specificity and rhamnosylation mechanism.     

Site-directed mutagenesis identifies catalytic residues in the active site of Escherichia coli phosphofructokinase.

Six active site mutants of Escherichia coli phosphofructokinase have been constructed and characterized using steady-state kinetics. All but one of the mutants (ES222) have significantly lower maximal activity, implicating these residues in the catalytic process. Replacement of Asp127, the key catalytic residue in the forward reaction with Glu, results in an enzyme with wild-type cooperative and allosteric behavior but severely decreased Fru6P binding. Replacement of the same residue with Tyr abolishes cooperativity while retaining sensitivity to allosteric inhibition and activation. Thus, this mutant has uncoupled homotropic from heterotropic allostery. Mutation of Asp103 to Ala results in an enzyme which retains wild-type Fru6P-binding characteristics with reduced activity. GDP, which allosterically activates the wild-type enzyme, acts as a mixed inhibitor for this mutant. Mutation of Thr125 to Ala and Asp129 to Ser produces mutants with impaired Fru6P binding and decreased cooperativity. In the presence of the activator GDP, both these mutants display apparent negative cooperativity. In addition, ATP binding is now allosterically altered by GDP. These results extend the number of active site residues known to participate in the catalytic process and help to define the mechanisms behind catalysis and homotropic and heterotropic allostery.     

The accessory Sec protein Asp2 modulates GlcNAc deposition onto the serine-rich repeat glycoprotein GspB

The accessory Sec system is a specialized transport system that exports serine-rich repeat (SRR) glycoproteins of Gram-positive bacteria. This system contains two homologues of the general secretory (Sec) pathway (SecA2 and SecY2) and several other essential proteins (Asp1 to Asp5) that share no homology to proteins of known function. In Streptococcus gordonii, Asp2 is required for the transport of the SRR adhesin GspB, but its role in export is unknown. Tertiary structure predictions suggest that the carboxyl terminus of Asp2 resembles the catalytic region of numerous enzymes that function through a Ser-Asp-His catalytic triad. Sequence alignment of all Asp2 homologues identified a highly conserved pentapeptide motif (Gly-X-Ser(362)-X-Gly) typical of most Ser-Asp-His catalytic triads, where Ser forms the reactive residue. Site-directed mutagenesis of residues comprising the predicted catalytic triad of Asp2 of S. gordonii had no effect upon GspB transport but did result in a marked change in the electrophoretic mobility of the protein. Lectin-binding studies and monosaccharide content analysis of this altered glycoform revealed an increase in glucosamine deposition. Random mutagenesis of the Asp2 region containing this catalytic domain also disrupted GspB transport. Collectively, our findings suggest that Asp2 is a bifunctional protein that is essential for both GspB transport and correct glycosylation. The catalytic domain may be responsible for controlling the glycosylation of GspB, while other surrounding regions are functionally required for glycoprotein transport.     

A theoretical study of the active sites of papain and S195C rat trypsin: implications for the low reactivity of mutant serine proteinases.

The serine and cysteine proteinases represent two important classes of enzymes that use a catalytic triad to hydrolyze peptides and esters. The active site of the serine proteinases consists of three key residues, Asp...His...Ser. The hydroxyl group of serine functions as a nucleophile and the imidazole ring of histidine functions as a general acid/general base during catalysis. Similarly, the active site of the cysteine proteinases also involves three key residues: Asn, His, and Cys. The active site of the cysteine proteinases is generally believed to exist as a zwitterion (Asn...His+...Cys-) with the thiolate anion of the cysteine functioning as a nucleophile during the initial stages of catalysis. Curiously, the mutant serine proteinases, thiol subtilisin and thiol trypsin, which have the hybrid Asp...His...Cys triad, are almost catalytically inert. In this study, ab initio Hartree-Fock calculations have been performed on the active sites of papain and the mutant serine proteinase S195C rat trypsin. These calculations predict that the active site of papain exists predominately as a zwitterion (Cys-...His+...Asn). However, similar calculations on S195C rat trypsin demonstrate that the thiol mutant is unable to form a reactive thiolate anion prior to catalysis. Furthermore, structural comparisons between native papain and S195C rat trypsin have demonstrated that the spatial juxtapositions of the triad residues have been inverted in the serine and cysteine proteinases and, on this basis, I argue that it is impossible to convert a serine proteinase to a cysteine proteinase by site-directed mutagenesis.     

Mutational analysis of a type II thioesterase associated with nonribosomal peptide synthesis.

Recent studies on type II thioesterases (TEIIs) involved in microbial secondary metabolism described a role for these enzymes in the removal of short acyl-S- phosphopantetheine intermediates from misprimed holo-(acyl carrier proteins) and holo-(peptidyl carrier proteins) of polyketide synthases and nonribosomal peptide synthetases. Because of the absence of structural information on this class of enzymes, we performed a mutational analysis on a prototype TEII essential for efficient production of the lipopeptide antibiotic surfactin (TEII(srf)), which led to identification of catalytic and structural residues. On the basis of sequence alignment of 16 TEIIs, 10 single and one double mutant of highly conserved residues of TEII(srf) were constructed and biochemically investigated. We clearly identified a catalytic triad consisting of Ser86, Asp190 and His216, suggesting that TEII(srf) belongs to the alpha/beta-hydrolase superfamily. Exchange of these residues with residues with aliphatic side chains abolished enzyme activity, whereas replacement of the active-site Ser86 with cysteine produced an enzyme with marginally reduced activity. In contrast, exchange of the second strictly conserved asparagine (Asp163) with Ala resulted in an active but unstable enzyme, excluding a role for this residue in catalysis and suggesting a structural function. The results define three catalytic and at least one structural residue in a nonribosomal peptide synthetase TEII.     

Gene cloning and biochemical characterization of eryngase,a serine aminopeptidase of Pleurotus eryngii belonging to the family S9 peptidases

Pleurotus eryngii serine aminopeptidase that has peptide bond formation activity, redesignated as eryngase, was cloned and expressed. Eryngase has a family S9 peptidase unit in the C-terminal region having a catalytic triad of Ser, Asp, and His. In the phylogenetic relations among the subfamilies of family S9 peptidase (S9A, prolyl oligopeptidase; S9B, dipeptidyl peptidase; S9C, acylaminoacyl peptidase; S9D, glutamyl endopeptidase), eryngase existed alone in the neighbor of S9C subfamily. Mutation of the active site Ser524 of the eryngase with Ala eliminated its catalytic activity. In contrast, S524C mutant maintained low catalytic activity. Investigation of aminolysis activity using l-Phe-NH₂ as a substrate showed that S524C mutant exhibited no hydrolysis reaction but synthesized a small amount of l-Phe-l-Phe-NH₂ by the catalysis of aminolysis. In contrast, wild-type eryngase hydrolyzed the product of aminolysis l-Phe-l-Phe-NH₂. Results show that the S524C mutant preferentially catalyzed aminolysis when on an l-Phe-NH₂ substrate.     

Functional topology of a surface loop shielding the catalytic center in lipoprotein lipase.

Lipoprotein lipase (LPL), hepatic lipase, and pancreatic lipase show high sequence homology to one another. The crystal structure of pancreatic lipase suggests that it contains a trypsin-like Asp-His-Ser catalytic triad at the active center, which is shielded by a disulfide bridge-bounded surface loop that must be repositioned before the substrate can gain access to the catalytic residues. By sequence alignment, the homologous catalytic triad in LPL corresponds to Asp156-His241-Ser132, absolutely conserved residues, and the homologous surface loop to residues 217-238, a poorly conserved region. To verify these assignments, we expressed in vitro wild-type LPL and mutant LPLs having single amino acid mutations involving residue Asp156 (to His, Ser, Asn, Ala, Glu, or Gly), His241 (to Asn, Ala, Arg, Gln, or Trp), or Ser132 (to Gly, Ala, Thu, or Asp) individually. All 15 mutant LPLs were totally devoid of enzyme activity, while wild-type LPL and other mutant LPLs containing substitutions in other positions were fully active. We further replaced the 22-residue LPL loop which shields the catalytic center either partially (replacing 6 of 22 residues) or completely with the corresponding hepatic lipase loop. The partial loop-replacement chimeric LPL was found to be fully active, and the complete loop-replacement mutant had approximately 60% activity, although the primary sequence of the hepatic lipase loop is quite different. In contrast, replacement with the pancreatic lipase loop completely inactivated the enzyme. Our results are consistent with Asp156-His241-Ser132 being the catalytic triad in lipoprotein lipase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Asparagine362 is essential for zinc binding and catalysis in the peptidase reaction of Saccharomyces cerevisiae leukotriene A4 hydrolase

Zinc metallopeptidases are ubiquitous enzymes with diverse cellular functions that can be found in most organisms. Leukotriene A₄ hydrolase (LTA4H; E.C. 3.3.2.6) is an unusual zinc metallopeptidase of the M1 family that also possesses an epoxide hydrolase activity; however, the role of its peptidase activity remains unknown. To further characterize the peptidase activity of LTA4H and other closely related metallopeptidases, a multiple sequence alignment and predicted structure were used to target three amino acid residues of yeast LTA4H for mutagenesis: Asn362, Trp365, and Asp399. Although mutating Trp365 and Asp399 had little effect on catalysis, altering Asn362 had varying effects on catalysis, depending on the replacement residue. Mutation of Asn362 to glutamine (N362Q) caused minor catalytic defects, while mutation to leucine (N362L) or glutamate (N362E) caused large reductions in activity. Both N362L and N362E also exhibited an altered pH dependence of catalysis, reduced chloride activation, and reduced zinc affinity and content, indicating that Asn362 may interact with the nearby zinc coordinating residue His344, and possibly with Glu363 as well, to polarize and/or orient these residues.     

Mutational analysis of Cvab, an ABC transporter involved in the secretion of active colicin V

CvaB is the central membrane transporter of the colicin V secretion system that belongs to an ATP-binding cassette superfamily. Previous data showed that the N-terminal and C-terminal domains of CvaB are essential for the function of CvaB. N-terminal domain of CvaB possesses Ca(2+)-dependent cysteine proteolytic activity, and two critical residues, Cys32 and His105, have been identified. In this study, we also identify Asp121 as being the third residue of the putative catalytic triad within the active site of the enzyme. The Asp121 mutants lose both their colicin V secretion activity and N-terminal proteolytic activity. The adjacent residue Pro122 also appears to play a critical role in the colicin V secretion. However, the reversal of the two residues D121P - P122D results in loss of activity. Based on molecular modeling and protein sequence alignment, several residues adjacent to the critical residues, Cys32 and His105, were also examined and characterized. Site-directed mutagenesis of Trp101, Asp102, Val108, Leu76, Gly77, and Gln26 indicate that the neighboring residues around the catalytic triad affect colicin V secretion. Several mutated CvaB proteins with defective secretion were also tested, including Asp121 and Pro122, and were found to be structurally stable. These results indicate that the residues surrounding the identified catalytic triad are functionally involved in the secretion of biologically active colicin V.     

Mutational analysis of the three cysteines and active-site aspartic acid 103 of ketosteroid isomerase from Pseudomonas putida biotype B.

In order to clarify the roles of three cysteines in ketosteroid isomerase (KSI) from Pseudomonas putida biotype B, each of the cysteine residues has been changed to a serine residue (C69S, C81S, and C97S) by site-directed mutagenesis. All cysteine mutations caused only a slight decrease in the k(cat) value, with no significant change of Km for the substrate. Even modification of the sulfhydryl group with 5,5'-dithiobis(2-nitrobenzoic acid) has almost no effect on enzyme activity. These results demonstrate that none of the cysteines in the KSI from P. putida is critical for catalytic activity, contrary to the previous identification of a cysteine in an active-site-directed photoinactivation study of KSI. Based on the three-dimensional structures of KSIs with and without dienolate intermediate analog equilenin, as determined by X-ray crystallography at high resolution, Asp-103 was found to be located within the range of the hydrogen bond to the equilenin. To assess the role of Asp-103 in catalysis, Asp-103 has been replaced with either asparagine (D103N) or alanine (D103A) by site-directed mutagenesis. For D103A mutant KSI there was a significant decrease in the k(cat) value: the k(cat) of the mutant was 85-fold lower than that of the wild-type enzyme; however, for the D103N mutant, which retained some hydrogen bonding capability, there was a minor decrease in the k(cat) value. These findings support the idea that aspartic acid 103 in the active site is an essential catalytic residue involved in catalysis by hydrogen bonding to the dienolate intermediate.     

Identification and analysis of a new family of bacterial serine proteinases

A family of hypothetical proteins, identified predominantly from archaeal genomes, has been analyzed in order to understand its functional characteristics. Using extensive sequence similarity searches it is inferred that this family is remotely related (best sequence identity is 19%) to ClpP proteinases that belongs to serine proteinase class. This family of hypothetical proteins is referred to as SDH proteinase family based on conserved sequential order of Ser, Asp and His residues and predicted serine proteinase activity. Results of fold recognition of SDH family sequences confirmed the remote relationship between SDH proteinases and Clp proteinases and revealed similar tertiary location of putative catalytic triad residues critical for serine proteinase function. However, the best sequence alignment we could obtain suggests that while catalytic Ser is conserved across Clp and SDH proteinases the location of the other catalytic triad residues, namely, His and Asp are swapped in their amino acid alignment positions and hence in 3-D structure. The evidence of conserved catalytic triad suggests that SDH could be a new family of serine proteinases with the fold of Clp proteinase, however sharing the catalytic triad order of carboxypeptidase clan. Signal peptide sequence identified at the N-terminus of some of the homologues suggests that these might be secretory serine proteinases involved in cleavage of extracellular proteins while the remote homologues, ClpP proteinases, are known to work in intracellular environment.     

Structural relationship between lipases and peptidases of the prolyl oligopeptidase family.

In prolyl oligopeptidase and its homologues, which constitute a new serine protease family, the order of the catalytic Ser and His residues in the amino acid sequence is the reverse of what is found in the trypsin and subtilisin families. The exact position of the third member of the catalytic triad, an Asp residue, has not yet been identified in the new family. Recent determination of the three-dimensional structures of pancreatic and microbial lipases has shown that the order of their catalytic residues is Ser, Asp, His, and this fits the order Ser, His of prolyl oligopeptidase. However, there is no sequence homology between lipases and peptidases, except for a 10-residue segment, which encompasses the essential Ser, and for the immediate vicinity of the catalytic Asp and His residues. This comparison identifies the catalytic Asp residue in the prolyl oligopeptidase family. The relative positions of the three catalytic residues in peptidases and microbial lipases were the same and this indicated structural and possibly evolutionary relationship between the two families.     

Function of the fully conserved residues Asp99, Tyr52 and Tyr73 in phospholipase A2

In the active centre of pancreatic phospholipase A2 His48 is at hydrogen-bonding distance to Asp99. This Asp-His couple is assumed to act together with a water molecule as a catalytic triad. Asp99 is also linked via an extended hydrogen bonding system to the side chains of Tyr52 and Tyr73. To probe the function of the fully conserved Asp99, Tyr52 and Tyr73 residues in phospholipase A2, the Asp99 residue was replaced by Asn, and each of the two tyrosines was separately replaced by either a Phe or a Gln. The catalytic and binding properties of the Phe52 and Phe73 mutants did not change significantly relative to the wild-type enzyme. This rules out the possibility that either one of the two Tyr residues in the wild-type enzyme can function as an acyl acceptor or proton donor in catalysis. The Gln73 mutant could not be obtained in any significant amounts probably due to incorrect folding. The Gln52 mutant was isolated in low yield. This mutant showed a large decrease in catalytic activity while its substrate binding was nearly unchanged. The results suggest a structural role rather than a catalytic function of Tyr52 and Tyr73. Substitution of asparagine for aspartate hardly affects the binding constants for both monomeric and micellar substrate analogues. Kinetic characterization revealed that the Asn99 mutant has retained no less than 65% of its enzymatic activity on the monomeric substrate rac 1,2-dihexanoyldithio-propyl-3-phosphocholine, probably due to the fact that during hydrolysis of monomeric substrate by phospholipase A2 proton transfer is not the rate-limiting step.(ABSTRACT TRUNCATED AT 250 WORDS)     

Site-directed mutagenesis of the putative catalytic triad of poliovirus 3C proteinase.

Based on predictions of the structure of proteinase 3C of poliovirus, mutations have been made at residues that are supposed to constitute the catalytic triad. Wild-type and mutant 3C were expressed in Escherichia coli, purified to homogeneity, and characterized by the ability to cleave a synthetic peptide substrate or an in vitro translated polypeptide consisting of part of the polyprotein of poliovirus. Additionally, the ability of autocatalytic processing of a precursor harboring wild-type or mutant 3C sequences was tested. Single substitutions of the residues His-40, Glu-71, and Cys-147 by Tyr, Gln, and Ser, respectively, resulted in an inactive enzyme. Replacement of Asp-85 by Asn resulted in an enzyme that was as active as wild-type enzyme in trans cleavage assays but whose autoprocessing ability was impaired. Our results are consistent with the proposal that residues His-40, Glu-71, and Cys-147 constitute the catalytic triad of poliovirus 3C proteinase. Furthermore, residue Asp-85 is not required for proper proteolytic activity despite being highly conserved between different picornaviruses. This indicates that Asp-85 might be involved in a different function of 3C.     

Structural Basis for Dimerization and Catalysis of a Novel Esterase from the GTSAG Motif Subfamily of the Bacterial Hormone-sensitive Lipase Family

Hormone-sensitive lipases (HSLs) are widely distributed in microorganisms, plants, and animals. Microbial HSLs are classified into two subfamilies, an unnamed new subfamily and the GDSAG motif subfamily. Due to the lack of structural information, the detailed catalytic mechanism of the new subfamily is not yet clarified. Based on sequence analysis, we propose to name the new subfamily as the GTSAG motif subfamily. We identified a novel HSL esterase E25, a member of the GTSAG motif subfamily, by functional metagenomic screening, and resolved its structure at 2.05 Å. E25 is mesophilic (optimum temperature at 50 °C), salt-tolerant, slightly alkaline (optimum pH at 8.5) for its activity, and capable of hydrolyzing short chain monoesters (C2–C10). E25 tends to form dimers both in the crystal and in solution. An E25 monomer contains an N-terminal CAP domain, and a classical α/β hydrolase-fold domain. Residues Ser¹⁸⁶, Asp²⁸², and His³¹² comprise the catalytic triad. Structural and mutational analyses indicated that E25 adopts a dimerization pattern distinct from other HSLs. E25 dimer is mainly stabilized by an N-terminal loop intersection from the CAP domains and hydrogen bonds and salt bridges involving seven highly conserved hydrophilic residues from the catalytic domains. Further analysis indicated that E25 also has some catalytic profiles different from other HSLs. Dimerization is essential for E25 to exert its catalytic activity by keeping the accurate orientation of the catalytic Asp²⁸² within the catalytic triad. Our results reveal the structural basis for dimerization and catalysis of an esterase from the GTSAG motif subfamily of the HSL family.     

Design of a serine protease-like catalytic triad on an antibody light chain displayed on the yeast cell surface

Lc-WT, the wild-type light chain of antibody, and Lc-Triad, its double mutant with E1D and T27aS designing for the construction of catalytic triad within Asp1, Ser27a, and original His93 residues, were displayed on the cell surface of the protease-deficient yeast strain BJ2168. When each cell suspension was reacted with BODIPY FL casein and seven kinds of peptide-MCA substrates, respectively, a remarkable difference in hydrolytic activities toward Suc-GPLGP-MCA (succinyl-Gly-Pro-Leu-Gly-Pro-MCA), a substrate toward collagenase-like peptidase, was observed between the constructs: Lc-Triad-displaying cells showed higher catalytic activity than Lc-WT-displaying cells. The difference disappeared in the presence of the serine protease inhibitor diisopropylfluorophosphate, suggesting that the three amino acid residues, Ser27a, His93, and Asp1, functioned as a catalytic triad responsible for the proteolytic activity in a similar way to the anti-vasoactive intestinal peptide (VIP) antibody light chain. A serine protease-like catalytic triad (Ser, His, and Asp) is considered to be directly involved in the catalytic mechanism of the anti-VIP antibody light chain, which moderately catalyzes the hydrolysis of VIP. These results suggest the possibility of new approach for the creation of tailor-made proteases beyond limitations of the traditional immunization approach.     

Identification of a catalytic residue of Clostridium paraputrificum N-acetyl-beta-D-glucosaminidase Nag3A by site-directed mutagenesis

Clostridium paraputrificum M-21 beta-N-acetylglucosaminidase 3A (Nag3A) is an enzyme classified in family 3 of the glycoside hydrolases. To identify catalytic residues of this enzyme, mutations were introduced into highly conserved Glu and Asp residues. Replacement of Asp175 with Ala abolished the catalytic activity without change in the circular dichroism spectrum, strongly suggesting that this residue is a catalytic residue, a nucleophile/base or a proton donor. Since the K(m) values of mutant enzymes D119N, D229N, D229A and D274N increased 17 to 41 times as compared with that of wild-type enzyme, Asp119, Asp229, and Asp274 appear to be involved in substrate recognition and binding. Taking previous studies into consideration, we presume that Asp303 is the catalytic nucleophile and Asp175 is the proton donor of C. paraputrificum Nag3A.     

Introduction of a free cysteinyl residue at position 68 in the subtilisin Savinase, based on homology with proteinase K.

Two subfamilies of the subtilisins, distinguished by the presence or absence of a free cysteinyl residue near the essential histidyl residue of the catalytic triad, are known. In order to evaluate the significance of the presence of this -SH group a cysteinyl residue has been introduced by site-directed mutagenesis into the cysteine-free subtilisin-like enzyme from Bacillus lentus, i.e. Savinase. The free cysteine affects the enzyme activity only slightly but renders it sensitive to mercurials presumably due to an indirect effect. The results indicate that the -SH group is not involved in catalysis.     

The catalytic site residues and interfacial binding of human pancreatic lipase.

In this study, the essential serine residue and 2 other amino acids in human pancreatic triglyceride lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) were tested for their contribution to the enzyme's catalytic site or interfacial binding site. By site-specific mutagenesis of the cDNA for human pancreatic lipase, amino acid substitutions were made at Ser153, His264, and Asp177. The mutant cDNAs were expressed in transfected COS-1 cells. Both the medium and the cells were examined for the presence of pancreatic lipase by Western blot analysis. The activity of the expressed proteins against triolein and the interfacial binding was measured. Proteins with mutations in Ser153 were secreted by the cells and bound to interfaces but had no detectable activity. Changing His264 to a leucine or Asp177 to an asparagine also produced inactive lipase. Substituting glutamic acid for Asp177 produced an active protein. These results demonstrate that Ser153 is involved in the catalytic site of pancreatic lipase and is not crucial for interfacial binding. Moreover, the essential roles of His264 and Asp177 in catalysis were demonstrated. A Ser-His-Asp catalytic triad similar to that present in serine proteases is present in human pancreatic lipase.