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G H de Haas

Publications and source records attributed to G H de Haas.

At least 19 recordsLinked to original sources
PubMed1998

Structural basis of the chiral selectivity of Pseudomonas cepacia lipase.

To investigate the enantioselectivity of Pseudomonas cepacia lipase, inhibition studies were performed with Sc- and Rc-(Rp,Sp)-1,2-dialkylcarbamoylglycero-3-O-p-nitrophenyl alkylphosphonates of different alkyl chain lengths. P. cepacia lipase was most rapidly inactivated by Rc-(Rp,Sp)-1,2-dioctylcarbamoylglycero-3-O-p-nitrophenyl octylphosphonate (Rc-trioctyl) with an inactivation half-time of 75 min, while that for the Sc-(Rp,Sp)-1,2-dioctylcarbamoylglycero-3-O-p-nitrophenyl octyl-phosphonate (Sc-trioctyl) compound was 530 min. X-ray structures were obtained of P. cepacia lipase after reaction with Rc-trioctyl to 0.29-nm resolution at pH 4 and covalently modified with Rc-(Rp,Sp)-1,2-dibutylcarbamoylglycero-3-O-p-nitrophenyl butyl-phosphonate (Rc-tributyl) to 0.175-nm resolution at pH 8.5. The three-dimensional structures reveal that both triacylglycerol analogues had reacted with the active-site Ser87, forming a covalent complex. The bound phosphorus atom shows the same chirality (Sp) in both complexes despite the use of a racemic (Rp,Sp) mixture at the phosphorus atom of the triacylglycerol analogues. In the structure of Rc-tributyl-complexed P. cepacia lipase, the diacylglycerol moiety has been lost due to an aging reaction, and only the butyl phosphonate remains visible in the electron density. In the Rc-trioctyl complex the complete inhibitor is clearly defined; it adopts a bent tuning fork conformation. Unambiguously, four binding pockets for the triacylglycerol could be detected: an oxyanion hole and three pockets which accommodate the sn-1, sn-2, and sn-3 fatty acid chains. Van der Waals' interactions are the main forces that keep the radyl groups of the triacylglycerol analogue in position and, in addition, a hydrogen bond to the carbonyl oxygen of the sn-2 chain contributes to fixing the position of the inhibitor.

Binding Sites↗
PubMed1995

Phosphonate analogues of triacylglycerols are potent inhibitors of lipase.

1,2-Dioctylcarbamoylglycero-3-O-p-nitrophenyl alkylphosphonates, with alkyl being methyl or octyl, were synthesised and tested as irreversible inhibitors of cutinase from Fusarium solani pisi and Staphylococcus hyicus lipase. Rapid inactivation of these enzymes occurred with a concomitant release of one mole of p-nitrophenol per mole of enzyme. With both lipases a higher reactivity was observed when the alkyl substituent on the phosphonate is a methyl rather than an octyl chain. Both lipases are highly selective for the chirality of these compounds at glycerol and at phosphorus. Rapid inactivation at an inhibitor concentration of 0.1 mol% in 100 mM NaTDOC (t 1/2 < 60 min.) occurred when the glycerol moiety had the (R) configuration, while inhibitors of the (S) configuration react 4-10-fold more slowly. The isomer with the p-nitrophenyl octylphosphonate attached to the secondary hydroxyl group of glycerol hardly inhibited (t 1/2 > 1 day) the lipases. These results reflect the known positional- and stereopreference of these enzymes which preferentially release the fatty acid at sn-3 of natural triacylglycerols. The enzymes appeared to be even more selective for the chirality at phosphorus, the differences in reactivity of the faster and slower reacting isomers being as high as about 250-fold for the methylphosphonates and about 60-fold for the octylphosphonates. These phosphonates can be regarded as true active site-directed inhibitors. The inhibited enzymes can be considered as analogues of the tetrahedral intermediate in the acylation step that occurs during triacylglycerol hydrolysis.

Carboxylic Ester Hydrolases↗
PubMed1995

Solution structure of porcine pancreatic phospholipase A2.

The lipolytic enzyme phospholipase A2 (PLA2) is involved in the degradation of high-molecular weight phospholipid aggregates in vivo. The enzyme has very high catalytic activities on aggregated substrates compared with monomeric substrates, a phenomenon called interfacial activation. Crystal structures of PLA2s in the absence and presence of inhibitors are identical, from which it has been concluded that enzymatic conformational changes do not play a role in the mechanism of interfacial activation. The high-resolution NMR structure of porcine pancreatic PLA2 free in solution was determined with heteronuclear multidimensional NMR methodology using doubly labeled 13C, 15N-labeled protein. The solution structure of PLA2 shows important deviations from the crystal structure. In the NMR structure the Ala1 alpha-amino group is disordered and the hydrogen bonding network involving the N-terminus and the active site is incomplete. The disorder observed for the N-terminal region of PLA2 in the solution structure could be related to the low activity of the enzyme towards monomeric substrates. The NMR structure of PLA2 suggests, in contrast to the crystallographic work, that conformational changes do play a role in the interfacial activation of this enzyme.

Animals↗
PubMed1995

Cutinase from Fusarium solani pisi hydrolyzing triglyceride analogues. Effect of acyl chain length and position in the substrate molecule on activity and enantioselectivity.

Triglyceride analogues were synthesized in which one of the primary acyl ester functions has been replaced by an alkyl group and the secondary acyl ester bond has been replaced by an acyl amino bond. The chain length at either position was varied, and both (R)- and (S)-enantiomers of each compound were synthesized. These pseudo triglycerides contain only one hydrolyzable ester bond, and they are ideally suited to studying the influence of the chain length at the 1-, 2-, and 3-position on lipase activity and on stereopreference. These substrates were used to characterize cutinase from Fusarium solani pisi. Our results show that the activity of cutinase is very sensitive to the length and distribution of the acyl chains and that the highest activities are found when the chains at positions 1 and 3 contain three or four carbon atoms. The enzyme preferentially hydrolyzes the (R)-enantiomers, but this preference is strongly dependent on the acyl chain length distribution, with (R) over (S) activity ratios varying from about 30 to 1. This enantioselectivity was found in three different assay systems: a mixed micellar, a reverse micellar, and a monolayer study. Our data suggest that at least two alkyl chains of the pseudo triglycerides must be fixed during hydrolysis. Therefore, these substrates were used to characterize mutants of cutinase with mutations in putative lipid binding domains. Two mutants (A85F and A85W) have increased activities. The results obtained with these mutants suggest an interaction of the acyl chain of the scissile ester bond with a surface loop, comprising residues 80-90, in the enzyme-substrate complex.

Carboxylic Ester Hydrolases↗
PubMed1995

Identification of the active site histidine in Staphylococcus hyicus lipase using chemical modification and mass spectrometry.

Staphylococcus hyicus lipase is a serine hydrolase. In order to identify the active site histidine of S. hyicus lipase we have chemically modified S. hyicus lipase with 1-bromo-octan-2-one. The enzyme is rapidly inactivated by this inhibitor with a half-time of 578 s at pH 6.5 and 30 degrees C. Addition of the enzyme's cofactor calcium increases the inactivation rate approx. 2-fold. When n-hexadecylphosphocholine, a non-hydrolysable substrate analogue, is added the inactivation rate decreases about 3-fold, suggesting that a residue in the active site of S. hyicus lipase is involved in the inactivation reaction. Inactivation of S. hyicus lipase with 14C-labelled 1-bromo-octan-2-one shows that 1.4 moles of inhibitor per mole of lipase are incorporated. The results of an electrospray mass spectrometric study of the inactivated enzyme are consistent with this finding. In order to identify the modified residue, both the inactivated and the unmodified lipase were digested with cyanogen bromide followed by trypsin. The resulting peptides were analysed using HPLC and fast atom bombardment mass spectrometry. The results allow the modified residue to be assigned to the peptide Gly597-Lys612. Collision induced dissociation mass spectrometry allowed the modified residue to be identified as His-600. From these results we conclude that this residue forms part of the catalytic triad of S. hyicus lipase.

Amino Acid Sequence↗
PubMed1995

Solution structure of porcine pancreatic phospholipase A2 complexed with micelles and a competitive inhibitor.

The three-dimensional structure of porcine pancreatic PLA2 (PLA2), present in a 40 kDa ternary complex with micelles and a competitive inhibitor, has been determined using multidimensional heteronuclear NMR spectroscopy. The structure of the protein (124 residues) is based on 1854 constraints, comprising 1792 distance and 62 phi torsion angle constraints. A total of 18 structures was calculated using a combined approach of distance geometry and restrained molecular dynamics. The atomic rms distribution about the mean coordinate positions for residues 1-62 and 72-124 is 0.75 +/- 0.09 A for the backbone atoms and 1.14 +/- 0.10 A for all atoms. The rms difference between the averaged minimized NMR structures of the free PLA2 and PLA2 in the ternary complex is 3.5 A for the backbone atoms and 4.0 A for all atoms. Large differences occur for the calcium-binding loop and the surface loop from residues 62 through 72. The most important difference is found for the first three residues of the N-terminal alpha-helix. Whereas free in solution Ala1, Leu2 and Trp3 are disordered, with the alpha-helical conformation with the alpha-amino group buried inside the protein. As a consequence, the important conserved hydrogen bonding network which is also seen in the crystal structures is present only in the ternary complex, but not in free PLA2. Thus, the NMR structure of the N-terminal region (as well as the calcium-binding loop and the surface loop) of PLA2 in the ternary complex resembles that of the crystal structure. Comparison of the NMR structures of the free enzyme and the enzyme in the ternary complex indicates that conformational changes play a role in the interfacial activation of PLA2.

Animals↗
PubMed1995

NMR structures of phospholipase A2 reveal conformational changes during interfacial activation.

It has long been proposed that the higher activity of phospholipase A2 (PLA2) for substrates presented as multimolecular aggregates compared to dispersed molecules (interfacial activation) arises due to a conformational change in the enzyme. X-ray studies have, however, failed to identify any such change. Here we report the solution structures of porcine pancreatic PLA2 both free and as a ternary complex with micelles and a competitive inhibitor. Important differences between these structures indicate that conformational changes may play an important role in the mechanism of interfacial activation in PLA2s.

Amino Acid Sequence↗
PubMed1994

Inactivation of Staphylococcus hyicus lipase by hexadecylsulfonyl fluoride: evidence for an active site serine.

The Staphylococcus hyicus lipase is an acyl hydrolase with broad substrate specificity including neutral glycerides and phospholipids. To obtain further insight into the mechanism of action of this enzyme, we tested several sulfonyl fluorides as active site-directed inhibitors. The enzyme is resistant to the well-known serine protease/esterase inhibitor phenylmethanesulfonyl fluoride (PMSF), but is rapidly inactivated by hexadecylsulfonyl fluoride. The kinetics of inactivation were studied in Triton X-100 micelles. Inactivation is fast and the rate of inactivation is constant over the pH range where this lipase is active. Metal ions like Ca2+ and Sr2+ do not appreciably influence the rate of inactivation, although the enzymatic activity is significantly increased, suggesting a structural role for these ions. The S. hyicus lipase contains a consensus sequence G-H/Y-S-X-G. Substitution by site-directed mutagenesis of this serine (Ser369) by a cysteine resulted in a mutant with only 0.2% residual activity. The activity of this mutant could not be inhibited with water-soluble sulfhydryl reagents either in the presence or absence of Triton X-100 micelles. In the presence of Triton X-100 micelles, inactivation of the mutant occurred with 4-nitrophenylhexadecyl disulfide (t1/2 = 125 min) while the wild-type enzyme does not react at all. We conclude that Ser369 is the active site residue and that in water this residue is inaccessible. Only after interfacial activation Ser369 (or Cys369) becomes exposed and reacts with irreversible inhibitors.

Bacterial Proteins↗
PubMed1993

Crystal structure of a porcine pancreatic phospholipase A2 mutant. A large conformational change caused by the F63V point mutation.

The highly homologous bovine and porcine pancreatic phospholipase A2 (85% amino acid residue identity) show a large conformational difference in the loop from residue 59 to 71. In bovine phospholipase A2 residues 59 to 66 adopt an alpha-helix conformation, while residues 67 to 71 are in a surface loop. Residues 59 to 66 in the porcine enzyme have a random coil conformation, and residues 67 to 71 form a short 3(10)-helix. It has been suggested that most probably this conformational difference is caused by the substitution Val63 (bovine) to Phe63 (porcine) in the otherwise invariant loop 59 to 70. To test this hypothesis, a mutant porcine phospholipase A2 was constructed in which residue Phe63 was replaced by a Val. The activity of this F63V mutant towards aggregated substrates was about half the activity of wild-type porcine phospholipase A2, but significantly different from that of the bovine enzyme. The affinity for zwitterionic interfaces was found to be intermediate between porcine and bovine phospholipase. The mutation did not have any effect on the stability of the enzyme towards denaturation by guanidine.HCl. The F63V mutant was crystallized in space group P2(1)2(1)2(1) with cell dimensions a = 79.88 A, b = 65.23 A, c = 52.62 A, with two molecules per asymmetric unit. Its three-dimensional structure was solved by molecular replacement methods, and refined to a crystallographic R-factor of 17.6% for all data between 10 and 2.2 A resolution. In one molecule the 58 to 71 loop is in very weak density, suggesting a high degree of disorder or flexibility. The conformation of the same loop in the other molecule could be determined unambiguously. It shows a conformation which resembles more that of bovine phospholipase A2 than that of porcine phospholipase. It is concluded that indeed the single F63V substitution causes a dramatic conformational change.

Amino Acid Sequence↗
PubMed1993

Competitive inhibition of lipolytic enzymes. IX. A comparative study on the inhibition of pancreatic phospholipases A2 from different sources by (R)-2-acylamino phospholipid analogues.

The inhibitory power (Z) of a number of (R)-1-alkyl-2-acylamino phospholipid analogues was determined for three mammalian phospholipases A2 from pig, ox and horse pancreas. All three enzymes display a clear preference for anionic (phosphoglycol) inhibitors over the zwitterionic (phosphocholine) derivatives; this effect is most pronounced for the bovine enzyme. Upon variation of the 1-alkyl chain length, the bovine and equine phospholipases, like the porcine enzyme in previous studies, show an optimum in Z for a six-carbon alkyl group. The introduction of a double bond in the 2-acylamino group generally improves the inhibitory power as compared with a fully saturated acyl chain. For the horse enzyme, the presence of an (R)-2-undecenoylamino group in the phosphocholine- and phosphoglycol-containing inhibitors resulted in affinities which are nearly 4 and 5 orders of magnitude higher, respectively, than for the substrate molecule. Direct determination of the dissociation constant Ki* of several inhibitors incorporated in a host lipid/water interface of non-inhibitory n-octadecenylphosphocholine micelles, was performed by ultraviolet difference spectroscopy. The progressive binding of a single inhibitor molecule into the active site of the three enzymes was followed quantitatively by an increasing tyrosine perturbation. With moderately strong competitive inhibitors (Z values ranging from about 50 to 10,000), quantitative values for Ki* were obtained. Extrapolation of the experimentally found linear relationship between Z and 1/Ki* yields predicted Ki* numbers for the much stronger inhibitors with Z values between 10,000 and 100,000.

Animals↗
PubMed1992

Conformational changes in phospholipase A2 upon binding to micellar interfaces in the absence and presence of competitive inhibitors. A 1H and 15N NMR study.

An NMR study has been made of porcine pancreatic phospholipase A2 (PLA) in three environments: free in solution, in a binary complex with dodecylphosphocholine micelles, and in a ternary complex with a micelle and the substrate-like inhibitor (R)-1-octyl-2-(N-dodecanoylamino)-2-deoxyglycero-3-phosph oglycol. 1H and 15N chemical shifts, amide exchange rates, and NOE intensities are compared for the enzyme in different environments. From these data, structural differences are found for the N-terminal part, the end of the surface loop at residues Tyr69 and Thr70, and the active site residue His48, and also for the Ca-binding loop (residues 28-32). Specifically, when binding to a micelle, the side chains of residues Ala1, Trp3, and Tyr69, as well as all protons of Thr70, are found to be closer together. After subsequent introduction of the competitive inhibitor, further changes are found for these residues. The N-terminus is flexible in PLA free in solution, in contrast with the crystal structures where it adopts an alpha-helical conformation. According to the NMR data, this helix is rigidly formed only in the ternary complex. Furthermore, in the ternary complex, the N-terminal amino group and the exchangeable hydrogen at N3 of the ring of His48 are observed. We propose that PLA is activated in two steps. An initial conformational change occurs upon binding to a micellar interface. The catalytically active conformation of the enzyme, which has an extensive network of hydrogen bonds, is formed only when binding a substrate or competitive inhibitor at a lipid-water interface.

Animals↗
PubMed1992

Competitive inhibition of lipolytic enzymes. VIII: Inhibitor-induced aggregation of porcine pancreatic phospholipase A2.

Several 2-acylaminophospholipid analogues have been demonstrated to behave as potent competitive inhibitors of porcine pancreatic phospholipase A2 (De Haas, G.H., Dijkman, R., Ransac, S. and Verger, R. (1990) Biochim. Biophys. Acta 1046, 249-257). Their inhibitory power appeared to be strictly controlled by the stereoconfiguration around the chiral C-2 atom and effective inhibition of the enzyme was observed only when incorporated into a micellar substrate-water interface. In the present study various direct binding techniques were applied to investigate the interaction of the enzyme with pure micelles of the stereoisomeric forms of 2-tetradecyl-amino-hexanol-1-phosphocholine (R-C14-PN and S-C14-PN). Upon equilibrium gel filtration of the enzyme (monomeric molecular mass = 14 kDa) on calibrated Superdex columns running in micellar solutions of R-C14-PN, the phospholipase eluted as a lipid-protein complex of 74 kDa. Under identical conditions, micellar solutions of S-C14-PN did not give rise to high-molecular mass aggregates and the enzyme eluted at its normal 14 kDa position. Light scattering experiments, ultrasedimentation and time-resolved fluorescence spectroscopy studies confirmed the formation of a high-molecular mass aggregate between enzyme and R-C14-PN micelles. The ultimate complex was shown to consist of four protein and about ten inhibitor molecules. Using time-resolved fluorescence spectroscopy the interaction was studied between the active site of phospholipase A2 and R-C14-PN molecules, both incorporated in an inert lipid matrix.

Animals↗
PubMed1992

Competitive inhibition of lipolytic enzymes. VII. The interaction of pancreatic phospholipase A2 with micellar lipid/water interfaces of competitive inhibitors.

In a recent series of kinetic studies (De Haas et al. (1990) Biochim. Biophys. Acta 1046, 249-257 and references therein) we have demonstrated that synthetic (R)-phospholipid analogues containing a 2-acylaminogroup instead of the 2-acyloxy function found in natural phospholipids, behave as strong competitive inhibitors of porcine pancreatic phospholipase A2 (PLA2). We also showed that these analogues strongly bind to the active site of the enzyme but only after their incorporation into a micellar substrate/water interface. In the present study we investigated the interaction of native PLA2 and of an inactive PLA2 in which the active site residue His-48 has been modified by alkylation with 1-bromo-2-octanone, with pure micelles of several of these inhibitors in both enantiomeric forms by means of ultraviolet difference absorption spectroscopy. Our results show that the first interaction step between native or modified enzyme and micellar lipid/water interfaces probably consists of a low-affinity Langmuir-type adsorption characterized by signals arising from the perturbation of the single Trp-3 residue. Once present at the interface the native enzyme is able to bind, in a second step, a single inhibitor molecule of the (R)-configuration in its active site, whereas the (S)-enantiomer is not bound in the active site. The overall dissociation constant of the interfacial phospholipase-inhibitor complex is three orders of magnitude lower for micelles composed of the (R)-isomer than those of the (S)-isomer. The modified PLA2 still adsorbs to micellar lipid/water interfaces but cannot bind either of the two enantiomers into its active site and similar dissociation constants were found for lipid-protein complexes with micelles of either the (R) or the (S) inhibitors. After blanking the ultraviolet signals due to the perturbation of Trp-3 in the initial adsorption step of the enzyme to a micellar surface of a non-inhibitory phospholipid analogue, the progressive binding of a single (R)-inhibitor molecule into the active site could be followed quantitatively by a tyrosine perturbation. These titrations yielded numerical values for the dissociation constants in the interface and provide a possible explanation for the large difference in overall dissociation constants of the complexes between enzyme and micelles of (R)-and (S)-inhibitors. With the use of PLA2 mutants in which each time a single tyrosine was replaced by phenylalanine, the tyrosine residues involved in binding of the monomeric inhibitor molecule were identified as Tyr-69 and Tyr-52.

Animals↗
PubMed1992

Rat platelet phospholipase A2. Kinetic characterization using the monomolecular film technique.

We have determined some kinetic parameters of rat platelet phospholipase A2, such as surface pressure dependency and substrate specificity, using the monomolecular film technique. We found that rat platelet phospholipase A2 is very specific for phospholipids having a negatively charged headgroup, no activity was detected when using zwitterionic phospholipids such as phosphatidylcholine. Furthermore, the interfacial pressure window which permits enzyme activity is very narrow as compared to pancreatic phospholipase A2. Maximal enzyme activity is found at 22 mN/m when using 1,2-dilauroylphosphatidylglycerol as substrate. Studies of the competitive inhibition of mixed films containing 2-acylaminophosphatidylglycol show that platelet phospholipase A2 is less sensitive than pancreatic and intestinal phospholipase A2. These results imply that, despite the high degree of sequence similarity, one must be very cautious in extrapolating inhibition data from one phospholipase A2 to similar enzymes from other origins.

Animals↗
PubMed1992

Competitive inhibition of lipolytic enzymes. VI. Inhibition of two human phospholipases A2 by acylamino phospholipid analogues.

The competitive inhibition of human pancreatic and a mutant human platelet phospholipase A2 (PLA2) was investigated using acylamino phospholipid analogues, which are potent competitive inhibitors of porcine pancreatic PLA2 [De Haas et al. (1990) Biochim. Biophys. Acta 1046, 249-257]. Both the mutant platelet PLA2 and the human pancreatic PLA2 are effectively inhibited by these compounds. The enzyme from platelets is most strongly inhibited by compounds with a negatively charged phosphoglycol headgroup. Compounds with a neutral phosphocholine headgroup are only weak inhibitors, whereas an inhibitor with a phosphoethanolamine headgroup shows an intermediate inhibitory capacity. The platelet PLA2 is most effectively inhibited by negatively charged inhibitors having a relatively short (four or more carbon atoms) alkylchain on position one and a acylamino chain of 14 carbon atoms on position two. For the pancreatic enzyme an inhibitor with a phosphoethanolamine headgroup was more effective than inhibitors with either a phosphocholine or a phosphoglycol headgroup. The chainlength preference of the pancreatic enzyme resembles that of the platelet PLA2. The largest discrimination in inhibition between the human platelet and the human pancreatic PLA2 is obtained with inhibitors with a negatively charged phosphoglycol headgroup, an alkyl chain of four carbon atoms on position one and a long acylamino chain of 14-16 carbon atoms on position two. Because the platelet PLA2 is thought to have several biological functions, specific inhibitors of this enzyme could have important implications in the design of pharmaceutically interesting compounds.

Binding, Competitive↗
PubMed1992

Site-directed mutagenesis and X-ray crystallography of two phospholipase A2 mutants: Y52F and Y73F.

Tyr52 and Tyr73 are conserved amino acid residues throughout all vertebrate phospholipases A2. They are part of an extended hydrogen bonding system that links the N-terminal alpha-NH3(+)-group to the catalytic residues His48 and Asp99. These tyrosines were replaced by phenylalanines in a porcine pancreatic phospholipase A2 mutant, in which residues 62-66 had been deleted (delta 62-66PLA2). The mutations did not affect the catalytic properties of the enzyme, nor the folding kinetics. The stability against denaturation by guanidine hydrochloride was decreased, however. To analyse how the enzyme compensates for the loss of the tyrosine hydroxyl group, the X-ray structures of the delta Y52F and delta Y73F mutants were determined. After crystallographic refinement the final crystallographic R-factors were 18.1% for the delta Y52F mutant (data between 7 and 2.3 A resolution) and 19.1% for the delta Y73F mutant (data between 7 and 2.4 A resolution). No conformational changes occurred in the mutants compared with the delta 62-66PLA2, but an empty cavity formed at the site of the hydroxyl group of the former tyrosine. In both mutants the Asp99 side chain loses one of its hydrogen bonds and this might explain the observed destabilization.

Enzyme Stability↗
PubMed1991

A monolayer and bulk study on the kinetic behavior of Pseudomonas glumae lipase using synthetic pseudoglycerides.

A heat-stable lipase from Pseudomonas glumae was purified to homogeneity. Its positional and stereospecific properties were investigated and compared with those of the well-known porcine pancreatic lipase. The kinetic properties of both enzymes were determined by use of six isomeric synthetic pseudoglycerides all composed of a single hydrolyzable fatty acyl ester bond and two lipase-resistant groups: one acylamino and one ether function. Two enzyme assay techniques were applied: a detergent-free system, the monomolecular surface film technique, and the pH-stat technique using clear micellar solutions of substrate in the presence of Triton X-100. Regarding the cleavage of primary ester bonds, P. glumae lipase possesses no stereopreference. In contrast, a large stereopreference in favor of the R-isomer is found for the hydrolysis of secondary ester bonds. Secondary ester bonds are efficiently cleaved by the lipase, which makes it of potential interest for enzymatic synthetic purposes. For the hydrolysis of this R-isomer a correlation between the experimental catalytic turnover rate and the binding constant for micelles was observed. The kinetic data of P. glumae lipase have been analyzed in terms of the scooting and hopping models for the action of lipolytic enzymes [Upreti, G.C., & Jain, M.K. (1980) J. Membr. Biol. 55, 113-121]. The results presented in this study are best explained by assuming that glumae lipase leaves the interface after a limited number of catalytic cycles.

Animals↗