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B Z Yu

Publications and source records attributed to B Z Yu.

At least 37 records · Page 2Linked to original sources
PubMed1995

Phospholipase A2 engineering. Probing the structural and functional roles of N-terminal residues with site-directed mutagenesis, X-ray, and NMR.

The N-terminal residues of phospholipase A2 (PLA2) are believed to be involved in the hydrogen-bonding network, the interfacial binding site, or the hydrophobic channel. Site-directed mutants of bovine pancreatic PLA2 with substitutions at positions 2, 3, 4, 5, 6, and 9 were constructed to test the roles of these residues in the structure and function of PLA2. Nonconservative mutations of Phe-5 and Ile-9, which are located inside the hydrophobic channel, led to significant perturbations in the conformation and conformational stability. Kinetic studies also indicated that mutations at Ile-9 and Phe-5 caused significant decreases in the rate of hydrolysis toward micellar and vesicle substrates. Scooting mode kinetic analysis showed that the binding step of the mutant enzymes to the DC14PM (1,2-dimyristoyl-sn-glycero-3-phosphomethanol) vesicle interface is not significantly affected and that the perturbations in catalysis occur mainly in kcat at the interface. The results taken together suggest that the residues Ile-9 and Phe-5 are important for both structure and catalysis. The mutant W3A (Trp-3 to Ala) also showed decreased rates of hydrolysis but to a lesser extent than Ile-9 and Phe-5 mutants. In addition, the binding affinity of W3A to the surface of the vesicles (i.e., the E to E* step) has been perturbed to the extent that hopping between anionic vesicles has been observed. On the other hand, the mutants of Gln-4 and Asn-6, which are located at or near the surface, displayed structural and kinetic properties similar to those of the wild-type PLA2 with the exception of the highly hydrophilic lysine mutant. The X-ray structure of the Q4E mutant indicates that the overall structure, the catalytic triad, and the link between residue 4 and Asp-99 via hydrogen bonding through Ala-1 and the structural water remain the same as in the WT. Substitutions for Leu at position 2 showed an acyl chain length discrimination toward different substrates, which may reflect the contacting position(s) of the substrate acyl chain with Leu-2.

Amino Acid Sequence↗
PubMed1995

Specific competitive inhibitor of secreted phospholipase A2 from berries of Schinus terebinthifolius.

Two structurally related triterpenoids 1 and 2 from pink peppercorn (berries of Schinus terebinthifolius) are identified and characterized as active site-directed specific competitive inhibitors of the three classes of secreted 14 kDa phospholipase A2. The inhibitors not only protect the active site histidine from alkylation but also inhibit the action of secreted phospholipase A2 from pig pancreas, human synovial fluid, and bee venom. Detailed X-ray crystallographic results on the structures of the inhibitors are provided. By physical methods and X-ray crystallography the triterpenoids were identified as masticadienoic acid and masticadienolic acid (schinol). Several other triterpenoids were ineffective as inhibitors of phospholipase A2; however certain ganoderic acid derivatives showed noticeable inhibition. Results show that the side chain of these acidic tetracyclic terpenoids can access the catalytic-site region of phospholipase A2, whereas the acyclic nucleus is at the interfacial recognition region. The selectivity of the assay protocol used here is demonstrated by the fact that the original screen of ethyl acetate extracts of 60 commercially available spices and herbs was carried out with phospholipase A2 from pig pancreas, and only one extract showed inhibitory action on the hydrolytic activity in the scooting mode. Under such assay conditions the enzyme remains tightly bound to the surface of the substrate vesicles. In this way, nonspecific effects of additives that promote desorption of the enzyme from the substrate vesicle surface, under conditions in which the binding of the enzyme to the vesicle is weak, are avoided. The assay protocol is useful for the kinetic characterization of the inhibitors of phospholipase A2, and it does not give false positive results with amphiphilic and hydrophobic compounds, as is the case with virtually all assay systems in use.

Animals↗
PubMed1994

Phospholipase A2 engineering. Structural and functional roles of the highly conserved active site residue aspartate-49.

Site-directed mutagenesis and high-resolution two-dimensional (2D) proton nuclear magnetic resonance (NMR) were used to probe the structural and functional roles of a highly conserved residue, Asp-49, in the interfacial catalysis by bovine pancreatic phospholipase A2 (PLA2, overexpressed in Escherichia coli). According to crystal structures, the side chain carboxylate of Asp-49, along with the carbonyl oxygens of Tyr-28, Gly-30, and Gly-32, and two water molecules, provides the necessary ligands for Ca2+ which is essential for the enzymatic activity. The Asp-49 of PLA2 was changed to Asn, Glu, Gln, Lys, and Ala; the resulting mutants are named D49N, D49E, D49Q, D49K, and D49A, respectively. The conformational stabilities of all five mutants are similar to that of WT as judged by guanidine hydrochloride-induced denaturation. The structural analyses by NMR indicated no global perturbations upon substitutions, although localized conformational perturbations can be observed for less conserved replacements. Direct Ca2+ binding studies showed no specific binding for D49A, D49N, D49Q, and D49K; however, D49E retains a 12-fold weaker calcium binding affinity (Kd,Ca = 23 mM). The specific activities of all five mutant enzymes decrease significantly, ranging from 5.4 x 10(2)- to 5.8 x 10(5)-fold in comparison with that of the wild-type enzyme. The observed activities of mutants require the presence of Ca2+. This demonstrates the functional importance of Asp-49 in the catalytic mechanism of PLA2, presumably by helping to bind and properly orient Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Functional significance of the conformational dynamics of the N-terminal segment of secreted phospholipase A2 at the interface.

The kinetic and fluorescence properties of several pig pancreatic phospholipase A2 (PLA2) with substitutions and deletions in the N-terminal region and of tyrosines 52 and 73 are characterized. The substitutions Ala-1-D-Ala or -Gly, Trp-3-Phe, Gln-4-Nle, Arg-6-Glu, Tyr-52-Phe, and Tyr-73-Phe had at the most only a modest effect on the interfacial catalytic activity on the anionic interface to which they bind with high affinity. The observed rate of hydrolysis in the scooting mode by deletion mutants lacking one or more successive residues from the N-terminal region was lower by 50-95%. Detailed kinetic analysis of the deletion mutant lacking Ala-1 (des-1-AMPA) showed that the 50% decrease in the rate is due to a 5-fold increase in the interfacial Michaelis-Menten parameter, KM*, without a significant change in kcat. These results and direct measurements show that the primary effect of Ala-1 deletion is to lower the affinity for the active site directed ligands. Although the affinity of these mutants for anionic interface remains the same as for the wild type, the affinity for zwitterionic neutral diluents is considerably lower. Significant differences in the fluorescence quantum yields and the heterogeneity in the frequency-domain fluorescence intensity decays of these enzymes suggest that both in solution and at the interface the N-terminal region is an ensemble of conformations rather than a discrete state. Additional results suggest that the interfacial microenvironment of Trp-3 in des-1-AMPA is more polar and Trp-3 is more accessible to quenching by acrylamide.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Relationship of interfacial equilibria to interfacial activation of phospholipase A2.

The equilibrium dissociation constants for the distribution of pig pancreatic phospholipase A2, its competitive inhibitors, and their complexes at the interface of a neutral diluent are determined. The relationship between these parameters and their significance for interfacial catalysis is elaborated in terms of a model based on the relationship between the underlying equilibria. By using a combination of spectroscopic and chemical modification methods, it was possible to determine the equilibrium dissociation constant of an inhibitor bound to the interface (K') or of the inhibitor bound to the enzyme in the aqueous phase (KI) or the interface (KI*). The equilibrium dissociation constant for the free enzyme (Kd) or for the enzyme-inhibitor complex (KdI) from the interface were also obtained. These constants are shown to be thermodynamically related, i.e., K'KdKI* = KIKdI, as predicted on the basis of the cyclic equilibrium scheme (thermodynamic box) describing the distribution of the enzyme and the inhibitor between the aqueous phase and the interface at constant calcium concentration. Results show that (i) calcium is required for the binding of a substrate or inhibitor molecule to the catalytic site; (ii) the effective dissociation constant of the inhibitor-enzyme complex in the aqueous phase is considerably larger than that for the enzyme at the interface, i.e., KI >> effective KI*; (iii) KdI does not depend on the structure of the inhibitor and Kd >> KdI; and (iv) structure-activity correlations suggest that ionic interactions between a ligand and the interfacial recognition site of the enzyme are important for K', which controls the concentration of the bound inhibitor that the enzyme "sees" in the interface. These observations demonstrate that the binding of the enzyme to the interface and the binding of the inhibitor to the active site of the enzyme at the interface are two distinguishable processes. Therefore, binding of a ligand to the active site of the enzyme promotes binding of the enzyme-inhibitor complex to other amphiphiles and the interface with higher affinity. It is suggested that the primary effect of binding the enzyme to the interface is to increase its intrinsic affinity toward the active-site-directed ligands, i.e., the interfacial activation of PLA2 is of K-type.

Animals↗
PubMed1993

The divalent cation is obligatory for the binding of ligands to the catalytic site of secreted phospholipase A2.

The divalent cation requirement for partial reactions of the catalytic turnover cycle during interfacial catalysis by pig pancreatic phospholipase A2 (PLA2) is investigated. Results show that the specific role of calcium in all the events of the catalytic cycle at the active site is not shared by other divalent cations. Cations such as calcium, barium, and cadmium bind to the enzyme in the aqueous phase. The active-site-directed ligands (substrate, products, and transition-state mimics) do not bind to the enzyme in the absence of a divalent cation. The synergistic binding of such ligands to the active site of PLA2 bound to the interface is, however, observed only in the presence of isosteric ions like calcium and cadmium, but not with larger ions like strontium or barium. The equilibrium constants for ligands bound to the enzyme in the presence of calcium and cadmium are virtually the same. However, only calcium supports the catalytic turnover; the rate of hydrolysis in the presence of cadmium is less than 1% of that observed with calcium. The role of divalent ions on the interfacial catalytic turnover cycle of PLA2 is not only due to the cation-assisted binding of the substrate but also due to its participation in the chemical step. Other roles of divalent ions in the events of interfacial catalytic turnover are also identified. For example, the binding of the enzyme to the interface is apparently promoted because the divalent cation is required for the sequential step, i.e., the binding of the substrate to the active site of PLA2.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Human nonpancreatic secreted phospholipase A2: interfacial parameters, substrate specificities, and competitive inhibitors.

The rate and equilibrium parameters for the interfacial catalysis by recombinant human nonpancreatic secreted phospholipase A2 were determined. Results show that the enzyme binds to anionic interfaces with considerably higher affinity than to zwitterionic interfaces. The extent of hydrolysis per enzyme on anionic vesicles in the processive scooting mode shows that the enzyme is fully catalytically active as a monomer. Among several secreted phospholipases A2 tested, the human nonpancreatic secreted enzyme is unique in its ability to undergo slow intervesicle exchange either by dissociation from the interface followed by binding to a different vesicle or by promoting the fusion of vesicles. The equilibrium dissociation constants for calcium, substrate analogs, reaction products, and several competitive inhibitors bound to the enzyme at the interface were determined by monitoring the ligand-conferred protection of the active site histidine residue from alkylation by phenacyl bromide. The interfacial Michaelis-Menten parameters were determined from the analysis of the entire reaction progress curve and also by monitoring the effect of competitive inhibitors on the initial rate of hydrolysis in the scooting mode. The interfacial Michaelis constant (KM*) for the substrate 1,2-dimyristoylglycero-sn-3-phosphomethanol was determined to be considerably above the maximal attainable mole fraction of unity for the substrate in the bilayer. Substrate specificity studies show that the enzyme does not significantly discriminate between phospholipids that differ in the type of polar head group or in the degree of unsaturation of the fatty acyl chains. Competitive inhibitors are described that display a high degree of selectivity for binding to the nonpancreatic versus pancreatic phospholipase A2. The kinetic properties of the human nonpancreatic secreted phospholipase A2 suggest that the enzyme has evolved to hydrolyze substrates at anionic interfaces and at high calcium concentrations.

Catalysis↗
PubMed1992

Phospholipase A2 engineering. The structural and functional roles of aromaticity and hydrophobicity in the conserved phenylalanine-22 and phenylalanine-106 aromatic sandwich.

The highly conserved phenylalanine-22 and phenylalanine-106, arranged as an aromatic sandwich, form part of an invariant hydrophobic wall that shields the active site of bovine pancreatic phospholipase A2 (PLA2) from bulk solvent [Dijkstra, B. W., Drenth, J., & Kalk, K. H. (1981) Nature 289, 604-606]. The residues have also been suggested to interact with the sn-2 acyl chain of bound phospholipid substrate [White, S. P., Scott, D. L., Otwinowski, Z., Gelb, M. H., & Sigler, P. B. (1990) Science 250, 1560-1563]. We now report the importance of these two residues in the structure and function of PLA2 in terms of aromaticity (changing to Ile) and hydrophobic (changing to Ala) and hydrophilic (changing to Tyr) character of these residues. The structural properties of the mutants were analyzed by proton NMR and by guanidine hydrochloride-induced denaturation. The functional properties were determined by measuring kinetic parameters toward various substrates in the forms of monomers, micelles, and vesicles, and by measuring equilibrium dissociation constants at the interface. The results show that (i) The conformational stability of each mutant was as good as that of wild-type PLA2; none of the mutants was significantly perturbed structurally as judged from detailed 1H NMR analysis. These results suggest that neither the Phe-22/Phe-106 face-to-face pair nor the Phe-22/Tyr-111 edge-to-face pair plays a significant structural role. (ii) Mutations to Ile at either position 22 or position 106 resulted in only minor perturbations in activity. This suggests that the aromaticity is not important to the function of these two residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1992

Interfacial catalysis by phospholipase A2: the rate-limiting step for enzymatic turnover.

The kinetics of the phospholipase A2-catalyzed hydrolysis of bilayer vesicles and mixed micelles of several oxyglycero and thioglycero analogues of phospholipids have been studied. The results with vesicles show that, depending on the source of the enzyme, the rates of hydrolysis of the oxy-containing long-chain phosphatidylmethanols are 2.5- to 28-fold higher compared to the rates of hydrolysis of the analogous thio substrates. The oxygen to sulfur substitution does not significantly alter the affinities of the enzymes for the reaction products or calcium. Since it is unlikely that sulfur substitution changes the rate constants for the formation and dissociation of the enzyme-product complex by the same factor, the element effects seen in the rates of hydrolysis of the oxy- and thioester phospholipids in vesicles are primarily due to a change in the rate constant for the chemical step of the catalytic turnover cycle. For bovine pancreatic phospholipase A2, various mutants with lower catalytic activity were used to show that the value of the element effect does not increase in the mutants. These results establish that, for the pancreatic phospholipase A2, the element effect is fully expressed, and the chemical step is fully rate-limiting for both oxyglycero and thioglycero phospholipids in vesicles. It was found that the element effect decreases from 7 to 1 when long-chain phosphatidylmethanols are present in micelles of a neutral diluent. This result suggests that the chemical step is not rate-limiting during the hydrolysis of these mixed micelle substrates.

Animals↗
PubMed1992

Phospholipase A2 engineering. Structural and functional roles of highly conserved active site residues tyrosine-52 and tyrosine-73.

Site-directed mutagenesis was used to probe the structural and functional roles of two highly conserved residues, Tyr-52 and Tyr-73, in interfacial catalysis by bovine pancreatic phospholipase A2 (PLA2, overproduced in Escherichia coli). According to crystal structures, the side chains of these two active site residues form H-bonds with the carboxylate of the catalytic residue Asp-99. Replacement of either or both Tyr residues by Phe resulted in only very small changes in catalytic rates, which suggests that the hydrogen bonds are not essential for catalysis by PLA2. Substitution of either Tyr residue by nonaromatic amino acids resulted in substantial decreases in the apparent kcat toward 1,2-dioctanoyl-sn-glycero-3-phosphocholine (DC8PC) micelles and the v(o) (turnover number at maximal substrate concentration, i.e., mole fraction = 1) toward 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DC14PM) vesicles in scooting mode kinetics [Berg, O. G., Yu, B.-Z., Rogers, J., & Jain, M. K. (1991) Biochemistry 30, 7283-7297]. The Y52V mutant was further analyzed in detail by scooting mode kinetics: the E to E* equilibrium was examined by fluorescence; the dissociation constants of E*S, E*P, and E*I (KS*, KP*, and KI*, respectively) in the presence of Ca2+ were measured by protection of histidine-48 modification and by difference UV spectroscopy; the Michaelis constant KM* was calculated from initial rates of hydrolysis in the absence and presence of competitive inhibitors; and the turnover number under saturating conditions (kcat, which is a theoretical value since the enzyme may not be saturated at the interface) was calculated from the vo and KM* values. The results indicated little perturbation in the interfacial binding step (E to E*) but ca. 10-fold increases in KS*, KP*, KI*, and KM* and a less than 10-fold decrease in kcat. Such changes in the function of Y52V are not due to global conformational changes since the proton NMR properties of Y52V closely resemble those of wild-type PLA2; instead, it is likely to be caused by perturbed enzyme-substrate interactions at the active site. Tyr-73 appears to play an important structural role. The conformational stability of all Tyr-73 mutants decreased by 4-5 kcal/mol relative to that of the wild-type PLA2. The proton NMR properties of Y73A suggested significant conformational changes and substantially increased conformational flexibility. These detailed structural and functional analyses represent a major advancement in the structure-function study of an enzyme involved in interfacial catalysis.

Animals↗
PubMed1992

Basis for the anomalous effect of competitive inhibitors on the kinetics of hydrolysis of short-chain phosphatidylcholines by phospholipase A2.

The effect of four specific competitive inhibitors on the kinetics of hydrolysis of short-chain diacyl-sn-glycero-3-phosphocholines below their critical micelle concentrations was examined. The kinetics of hydrolysis of short-chain substrates dispersed as solitary monomers were generally consistent with the classical Michaelis-Menten formalism; i.e., hydrolysis began without any latency period, the steady-state rate was observed at higher substrate concentrations, the steady-state initial rate showed a linear dependence on the enzyme concentration, and the hyperbolic dependence of the initial rate on the substrate concentration could be described in terms of KM and Vmax parameters. The competitive nature of the inhibitors used in this study has been established by a variety of techniques, and the equilibrium dissociation constants for the inhibitors bound to the enzyme were measured by the protection method [Jain et al. (1991) Biochemistry 30, 7306-7317]. The kinetics of hydrolysis in the presence of competitive inhibitors could be described by a single dissociation constant. However, the value of the dissociation constant obtained under the kinetic conditions was comparable to that obtained by the protection method for the inhibitor-enzyme complex bound to a neutral diluent, rather than to the value of the dissociation constant obtained with solitary monomeric inhibitors and the enzyme in the aqueous phase. Spectroscopic methods showed that the effectively lower dissociation constant of an inhibitor bound to PLA2 at the interface is due to the stabilization of the enzyme-inhibitor complex by interaction with other amphiphiles present in the reaction mixture.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1992

[Evaluation of the treatment of adult tuberculous meningitis].

Through analysis of the treatment in 205 patients of adult tuberculous meningitis, we considered: 1. effective and vigorous alleviation of high intracranial pressure in early stage, 2. appropriate dosage of corticosteroids and, 3. correct selection of antituberculous drugs was the mainstay for chemotherapy of tuberculous meningitis. The results were 82 patients (40.0%) were cured, 101 cases (49.3%) improved, 15 cases (7.3%) died and 7 cases (3.4%) had complications. 175 cases have been followed-up from two to five years, all of them are alive.

Adolescent↗
PubMed1991

Active-site-directed specific competitive inhibitors of phospholipase A2: novel transition-state analogues.

More than 100 amphiphilic phosphoesters, possible tetrahedral transition-state analogues capable of coordinating to the calcium ion at the active site of phospholipase A2, were designed, synthesized, and tested as inhibitors for the hydrolysis of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol vesicles in the scooting mode. This assay system permits the study of structurally diverse inhibitors with phospholipase A2S from different sources, and it is not perturbed by factors that change the quality of the interface. As a prototype, 1-hexadecyl-3-trifluoroethylglycero-2-phosphomethanol (MJ33) was investigated in detail. Only the (S)-(+) analogue of MJ33 is inhibitory, and it is as effective as the sn-2 phosphonate or the sn-2 amide analogues of sn-3 phospholipids. The inhibitory potencies of the various phosphoesters depended strongly on the stereochemical and structural features, and the mole fractions of inhibitors required for 50% inhibition, X1(50), ranged from more than 1 to less than 0.001 mole fraction. The affinity of certain inhibitors for enzymes from different sources differed by more than 200-fold. The inhibitors protected the catalytic site residue His-48 from alkylation in the presence of calcium but not barium as expected if the formation of the EI complex is supported only by calcium. The equilibrium dissociation constant for the inhibitor bound to the enzyme at the interface was correlated with the XI(50) values, which were different if the inhibition was monitored in the pseudo-zero-order or the first-order region of the progress curve. These results show that the inhibitors described here interfered only with the catalytic turnover by phospholipase A2's bound to the interface, their binding to the enzyme occurred through calcium, and the inhibitors did not have any effect on the dissociation of the enzyme bound to the interface.

Animals↗
PubMed1991

Kinetic characterization of phospholipase A2 modified by manoalogue.

Manoalogue, a synthetic analogue of the sea sponge-derived manoalide, has been previously shown to partially inactivate the phospholipase A2 from cobra venom (Reynolds, L. J., Morgan, B. P., Hite, E. D., Mihelich, E. D., & Dennis, E. A. (1988) J. Am. Chem. Soc. 110, 5172) by reacting with enzyme lysine residues. In the present study, the inactivation of the phospholipases A2 from pig pancreas, bee venom, and cobra (Naja naja naja) venom by manoalogue was studied in detail. Manoalogue-treated enzymes were examined in the scooting mode on vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol. Here the native enzymes bound irreversibly to the vesicles and hydrolyzed all of the phospholipids in the outer monolayer without leaving the surface of the interface. All three manoalogue-treated enzymes showed reduced catalytic turnover for substrate hydrolysis in the scooting mode, and the modified enzymes did not hop from one vesicle to another. Thus, inactivation by manoalogue is not due to the decrease in the fraction of enzyme bound to the substrate interface. This result was also confirmed by fluorescence studies that directly monitored the binding of phospholipase A2 to vesicles. A chemically modified form of the pig pancreatic phospholipase A2 in which all of the lysine epsilon-amino groups have been amidinated was not inactivated by manoalogue, indicating that the modification of lysine residues and not the amino-terminus is required for the inactivation. Several studies indicated that the manoalogue-modified enzymes contain a functional active site. For example, studies that monitored the protection by ligands of the active site from attack by a alkylating agent showed that manoalogue-modified pig phospholipase A2 was capable of binding calcium, a substrate analogue, lipolysis products, and a competitive inhibitor. Furthermore, relative to native enzymes, manoalogue-modified enzymes retained significantly higher catalytic activities when acting on water-soluble substrates than when acting on vesicles in the scooting mode. Intact manoalogue had no affinity for the catalytic site on the enzyme as it did not inhibit the enzyme in the scooting mode and it did not protect the active site from alkylation. Pig pancreatic phospholipase A2 bound to micelles of 2-hexadecyl-sn-glycero-3-phosphocholine was resistant to inactivation by manoalogue, suggesting that the modification of lysine residues on the interfacial recognition surface of the enzyme was required for inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Interfacial catalysis by phospholipase A2: determination of the interfacial kinetic rate constants.

Hydrolysis of vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DMPM) by pig pancreatic phospholipase A2 (PLA2) occurs in a highly processive "scooting" mode, and the rate is comparable to or exceeds the rates observed with detergent-dispersed mixed micelles under optimal conditions. A complete kinetic description of the steady-state time course of the hydrolysis is developed. The analysis covers the whole Michaelis-Menten space: it emphasizes the key features of interfacial catalysis by a detailed theoretical analysis, describes the experimental protocols to determine the values of the kinetic and equilibrium constants for interfacial catalysis, and provides an interpretation of the effect of calcium, substrate, products, apparent activators, and competitive inhibitors on the reaction progress curve by a single set of rate and equilibrium parameters. In this paper, the integrated reaction progress curve was rigorously interpreted in terms of a minimal model involving the Michaelis-Menten reaction sequence in the interface: E* + S in equilibrium E*S----E*P in equilibrium E* + P, and most of the individual rate and equilibrium constants for the catalytic cycle were determined. This rigorous description of interfacial catalysis was made experimentally possible by examining the action of PLA2 in the scooting mode under conditions of at most one enzyme per vesicle, where it hydrolyzed all of the substrate in the outer monolayer of vesicles without leaving the surface. Other experimentally verified constraints for this analysis include the following: all enzyme was bound to vesicles; the integrity of vesicles was maintained during the course of hydrolysis; and the substrate, enzyme, and products did not exchange between vesicles nor did they exchange across the bilayer. The mechanistic significance of the rate constants is discussed in the accompanying papers.

Animals↗
PubMed1991

Interfacial catalysis by phospholipase A2: dissociation constants for calcium, substrate, products, and competitive inhibitors.

Interpretation of the kinetics of interfacial catalysis in the scooting mode as developed in the first paper of this series [Berg et al. (1991) Biochemistry 30 (first paper of six in this issue)], was based on the binding equilibrium for a ligand to the catalytic site of phospholipase A2. In this paper, we describe direct methods to determine the value of the Michaelis-Menten constant (KMS) for the substrate, as well as the equilibrium dissociation constants for ligands (KL) such as inhibitors (KI), products (KP), calcium (KCa), and substrate analogues (KS) bound to the catalytic site of phospholipase A2 at the interface. The KL values were obtained by monitoring the susceptibility to alkylation of His-48 at the catalytic site of pig pancreatic PLA2 bound to micellar dispersions of the neutral diluent 2-hexadecyl-sn-glycero-3-phosphocholine. The binding of the enzyme to dispersions of this amphiphile alone had little effect on the inactivation rate. The half-time for inactivation of the enzyme bound to micelles of the neutral diluent depended not only on the nature of the alkylating agent but also on the structure and the mole fraction of other ligands at the interface. The KL values for ligands obtained from the protection studies were in excellent accord with those obtained by monitoring the activation or inhibition of hydrolysis of vesicles of 1,2-dimyristoyl-sn-glycerophosphomethanol. Since only calcium, competitive inhibitors, and substrate analogues protected phospholipase A2 from alkylation, this protocol offered an unequivocal method to discern active-site-directed inhibitors from nonspecific inhibitors of PLA2, such as local anesthetics, phenothiazines, mepacrine, peptides related to lipocortin, 7,7-dimethyleicosadienoic acid, quinacrine, and aristolochic acid, all of which did not have any effect on the kinetics of alkylation nor did they inhibit the catalysis in the scooting mode.

Animals↗
PubMed1991

Interfacial catalysis by phospholipase A2: substrate specificity in vesicles.

The binding equilibrium of phospholipase A2 (PLA2) to the substrate interface influences many aspects of the overall kinetics of interfacial catalysis by this enzyme. For example, the interpretation of kinetic data on substrate specificity was difficult when there was a significant kinetic contribution from the interfacial binding step to the steady-state catalytic turnover. This problem was commonly encountered with vesicles of zwitterionic phospholipids, where the binding of PLA2 to the interface was relatively poor. The action of PLA2 on phosphatidylcholine (PC) vesicles containing a small amount of anionic phospholipid, such as phosphatidic acid (PA), was studied. It was shown that the hydrolysis of these mixed lipid vesicles occurs in the scooting mode in which the enzyme remains tightly bound to the interface and only the substrate molecules present on the outer monolayer of the target vesicle became hydrolyzed Thus the phenomenon of scooting mode hydrolysis was not restricted to the action of PLA2 on vesicles of pure anionic phospholipids, but it was also observed with vesicles of zwitterionic lipids as long as a critical amount of anionic compound was present. Under such conditions, the initial rate of hydrolysis of PC in the mixed PC/PA vesicles was enhanced more than 50-fold. Binding studies of PLA2 to vesicles and kinetic studies in the scooting mode demonstrated that the enhancement of PC hydrolysis in the PC/PA covesicles was due to the much higher affinity of the enzyme toward covesicles compared to vesicles of pure PC phospholipids. A novel and technically simple protocol for accurate determination of the substrate specificity of PLA2 at the interface was also developed by using a double-radiolabel approach. Here, the action of PLA2 in the scooting mode was studied on vesicles of the anionic phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphomethanol that contained small amounts of 3H- and 14C-labeled phospholipids. From an analysis of the 3H and 14C radioactivity in the released fatty acid products, the ratio of substrate specificity constants (kcat/KMS) was obtained for any pair of radiolabeled substrates. These studies showed that the PLA2s from pig pancreas and Naja naja naja venom did not discriminate between phosphatidylcholine and phosphatidylethanolamine phospholipids or between phospholipids with saturated versus unsaturated acyl chains and that the pig enzyme had a slight preference for anionic phospholipids (2-3-fold). The described protocol provided an accurate measure of the substrate specificity of PLA2 without complications arising from the differences in binding affinities of the enzyme to vesicles composed of pure phospholipids.

Animals↗