The affinity of phospholipase A2 for the interface of the substrate and analogs

In the intravesicle scooting mode of interfacial catalysis, the interfacial complex E*S is formed by the interaction of the membrane bound phospholipase A2 (E*) with the substrate monomer (S) in the interface. In the presence of nonhydrolyzable substrate analogs (I) the kinetics of interfacial catalysis is modified. If phospholipase A2 is added to a mixture of the vesicles of L-DMPMe ester and of DTPMe ether or D-DMPMe ester, the extent of hydrolysis, A, decreases and the interfacial scooting rate constant, ki, remains unchanged. On the other hand, when the enzyme is added to the vesicles prepared from premixed L-DMPMe ester with D-DMPMe ester or L-DTPMe ether, ki decreases but A remains constant. Qualitatively, these results are in excellent accord with the Scheme I for interfacial catalysis. However, a quantitative departure has been noted, which suggests that the interfacial dissociation constant for E*S is larger than that for E*I. These results are interpreted to suggest that the catalytic rate constant for decomposition of E*S to E* + P is larger than the rate constant for decomposition of E*S to E* + S. Broader implications of the scooting mode of interfacial catalysis are discussed.     

Interfacial catalysis by phospholipase A2: evaluation of the interfacial rate constants by steady-state isotope effect studies.

The kinetics of hydrolysis of phospholipid vesicles by phospholipase A2 (PLA2) in the scooting mode can be described by the Michaelis-Menten formalism for the action of the enzyme in the interface (E*). E* + S in equilibrium E*S in equilibrium E*P in equilibrium E* + Products The values of the interfacial rate constants cannot be obtained by classical methods because the concentration of the substrate within the lipid bilayer is not easily manipulated. In the present study, carbonyl-carbon heavy atom isotope effects for the hydrolysis of phospholipids have been measured in both vesicles and in mixed micelles in which the phospholipid was present in the nonionic detergent Triton X-100. A large [14C]carbonyl carbon isotope effect of 1.12 +/- 0.02 was measured for the cobra venom PLA2-catalyzed hydrolysis of dipalmitoylphosphatidylcholine in Triton X-100. In contrast, no isotope effect (1.01 +/- 0.01) was measured for the action of the porcine pancreatic and cobra venom enzymes on vesicles of dimyristoylphosphatidylmethanol in the scooting mode. In a second experiment, the hydrolysis of vesicles was carried out in oxygen-18 enriched water. Analysis of the released fatty acid product by mass spectrometry showed that it contained only a single oxygen-18. All of these results were used to estimate both the forward and reverse commitments to catalysis. The lack of doubly labeled fatty acid demonstrated that the product is released from the E*P complex faster than the reverse of the esterolysis step. The small isotope effect in vesicles demonstrated that the E*S complex goes on to products faster than substrate is released from the enzyme. The relevance of these results to an understanding of substrate specificity and inhibition of PLA2 is discussed. In addition, the conditions placed on the values of the rate constants obtained in the present study together with results obtained in the other studies described in this series of papers have led to the evaluation of most of the interfacial rate constants for the hydrolysis of phospholipid vesicles by PLA2.     

Contributions of residues of pancreatic phospholipase A2 to interfacial binding, catalysis, and activation

Primary rate and equilibrium parameters for 60 site-directed mutants of bovine pancreatic phospholipase A2 (PLA2) are analyzed so incremental contributions of the substitution of specific residues can be evaluated. The magnitude of the change is evaluated so a functional role in the context of the N- and C-domains of PLA2 can be assigned, and their relationship to the catalytic residues and to the i-face that makes contact with the interface. The effect of substitutions and interfacial charge is characterized by the equilibrium dissociation constant for dissociation of the bound enzyme from the interface (Kd), the dissociation constant for dissociation of a substrate mimic from the active site of the bound enzyme (KL), and the interfacial Michaelis constants, KM and kcat. Activity is lost (>99.9%) on the substitution of H48 and D49, the catalytic residues. A more than 95% decrease in kcat is seen with the substitution of F5, I9, D99, A102, or F106, which form the substrate binding pocket. Certain residues, which are not part of the catalytic site or the substrate binding pocket, also modulate kcat. Interfacial anionic charge lowers Kd, and induces kcat activation through K56, K53, K119, or K120. Significant changes in KL are seen by the substitution of N6, I9, F22, Y52, K53, N71, Y73, A102, or A103. Changes in KM [=(k2+k-1)/k1] are attributed to kcat (=k2) and KL (=k-1/k1). Some substitutions change more than one parameter, implying an allosteric effect of the binding to the interface on KS, and the effect of the interfacial anionic charge on kcat. Interpreted in the context of the overall structure, results provide insights into the role of segments and domains in the microscopic events of catalytic turnover and processivity, and their allosteric regulation. We suggest that the interfacial recognition region (i-face) of PLA2, due to the plasticity of certain segments and domains, exercises an allosteric control on the substrate binding and chemical step.     

Interfacial catalysis by phospholipase A2: activation by substrate replenishment.

Polymyxin B (Px), a cyclic cationic peptide, was shown to act as a potent activator of interfacial catalysis by phospholipase A2 (PLA2) acting on dimyristoylphosphatidylmethanol vesicles in the scooting mode. A 7-fold increase in the initial enzymatic velocity was seen with the pig pancreatic PLA2 in the presence of 1 microM Px. Initial experiments including the dependency of the degree of activation by Px on the source of the PLA2 suggested that Px bound to a cationic binding site on the enzyme. However, numerous additional observations led to the conclusion that activation by Px was due to its effects on the substrate interface. For example, the activation by Px was only seen when the PLA2 acted on small vesicles rather than larger ones, and all of the available substrate was eventually hydrolyzed in the presence of a small mole fraction of Px. Px did not promote the intervesicle exchange of PLA2, and it did not alter the binding of the evidence led to the conclusion that Px activated interfacial catalysis by promoting the replenishment of substrate in the enzyme-containing vesicles. When PLA2 was acting on small vesicles in the scooting mode, the observed initial velocity was lower than that measured with large vesicles because the surface concentration of substrate decreased relatively rapidly in the small vesicles. Px promoted the transfer of phospholipids between the vesicles and functioned as an activator by keeping the mole fraction of substrate in the enzyme-containing vesicles close to 1. This effect of Px was consistent with the ability of polycationic peptides to induce the intervesicle mixing of anionic phospholipids in vesicles [Bondeson, J., & Sundler, R. (1990) Biochim. Biophys. Act 1026, 186-194]. Activation by substrate replenishment was quantitatively predicted by the theory of interfacial catalysis on vesicles in the scooting mode. The role of substrate replenishment in the kinetics of interfacial catalysis in phospholipid micelles was discussed. Finally, the protocols developed in this paper were outlined in view of their utility in the analysis of activators of interfacial catalysis.     

Phospholipase A2 engineering. X-ray structural and functional evidence for the interaction of lysine-56 with substrates.

Site-directed mutagenesis studies of bovine pancreatic phospholipase A2 (PLA2, overproduced in Escherichia coli) showed that replacement of surface residue Lys-56 by a neutral or hydrophobic amino acid residue resulted in an unexpected and significant change in the function of the enzyme. The kcat for phosphatidylcholine micelles increases 3-4-fold for K56M, K56I, and K56F and ca. 2-fold for K56N and K56T but does not change for K56R. These results suggest that the side chain of residue 56 has significant influence on the activity of PLA2. In order to probe the structural basis for the enhanced activity, the crystal structures of wild-type and K56M PLA2 were determined by X-ray crystallography to a resolution of 1.8 A. The results suggest that the mutation has not only perturbed the conformation of the side chain of Met-56 locally but also caused conformational changes in the neighboring loop (residues 60-70), resulting in the formation of a hydrophobic pocket by residues Met-56, Tyr-52, and Tyr-69. Docking of a phosphatidylcholine inhibitor analogue into the active site of K56M, according to the structure of the complex of cobra venom PLA2-phosphatidylethanolamine inhibitor analogue [White, S.P., Scott, D. L., Otwinowski, Z., Gleb, M. H., & Sigler, P. (1990) Science 250, 1560-1563], showed that the choline moiety [N(CH3)3]+ is readily accommodated into the newly formed hydrophobic pocket with a high degree of surface complementarity. This suggests a possible interaction between residue 56 and the head group of the phospholipid, explaining the enhanced activities observed when the positively charged Lys-56 is substituted by apolar residues, viz., K56M, K56I, and K56F. Further support for this interpretation comes from the 5-fold enhancement in kcat for the mutant K56E with a negatively charged side chain, where there would be an attractive electrostatic interaction between the side chain of Glu-56 and the positively charged choline moiety. Our results also refute a recent report [Tomasselli, A. G., Hui, J., Fisher, J., Zürcher-Neely, H., Reardon, I.M., Oriaku, E., Kézdy, F.J., & Heinrikson, R.L. (1989) J. Biol. Chem. 264, 10041-10047] that substrate-level acylation of Lys-56 is an obligatory step in the catalysis by PLA2.     

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.     

The functional role of tyrosine-200 in human testis angiotensin-converting enzyme.

The active site of angiotensin-converting enzyme (ACE) has been shown by chemical modification to contain a critical tyrosine residue, identified as Tyr-200 in human testis ACE (hTACE). We have expressed a mutant hTACE containing a Tyr-200 to Phe mutation. The mutant exhibits a marked decrease in kcat: 15-fold and 7-fold for the hydrolysis of furanacryloyl-Phe-Gly-Gly and angiotensin I, respectively, whereas its Km increases by only 1.6- and 2.2-fold, respectively. We conclude that Tyr-200 is not required for substrate binding. Instead, the effect on kcat together with a 100-fold decrease in affinity for the ACE inhibitor lisinopril indicates that Tyr-200 may participate in catalysis by stabilizing the transition state complex. Thus, Tyr-200 in hTACE has a role analogous to that of Tyr-198 in carboxypeptidase A.     

Assay of phospholipases A(2) and their inhibitors by kinetic analysis in the scooting mode

Several cellular processes are regulated by interfacial catalysis on biomembrane surfaces. Phospholipases A(2) (PLA(2)) are interesting not only as prototypes for interfacial catalysis, but also because they mobilize precursors for the biosynthesis of eicosanoids and platelet activating factor, and these agents ultimately control a wide range of secretory and inflammatory processes. Since PLA(2) carry out their catalytic function at membrane surfaces, the kinetics of these enzymes depends on what the enzyme 'sees' at the interface, and thus the observed rate is profoundly influenced by the organization and dynamics of the lipidwater interface ('quality of the interface'). In this review we elaborate the advantages of monitoring interfacial catalysis in the scooting mode, that is, under the conditions where the enzyme remains bound to vesicles for several thousand catalytic turnover cycles. Such a highly processive catalytic turnover in the scooting mode is useful for a rigorous and quantitative characterization of the kinetics of interfacial catalysis. This analysis is now extended to provide insights into designing strategy for PLA(2) assays and screens for their inhibitors.     

Pseudomonas aeruginosa exotoxin A: effects of mutating tyrosine-470 and tyrosine-481 to phenylalanine

Directed mutagenesis was used to probe the functions of Tyr-470 and Tyr-481 of Pseudomonas aeruginosa exotoxin A (ETA) with respect to cytotoxicity, ADP-ribosylation of elongation factor 2 (EF-2), and NAD-glycohydrolase activity. Both of these residues lie in the active site cleft, close to Glu-553, a residue believed to play a direct role in catalysis of ADP-ribosylation of EF-2. Substitution of Tyr-470 with Phe caused no change in any of these activities, thus eliminating the possibility that the phenolic hydroxyl group of Tyr-470 might be directly involved in catalysis. Mutation of Tyr-481 to Phe caused an approximately 10-fold reduction in NAD:EF-2 ADP-ribosyltransferase activity and cytotoxicity but no change in NAD-glycohydrolase activity. The latter mutation did not alter the KM of NAD in the NAD-glycohydrolase reaction, which suggests that the phenolic hydroxyl of Tyr-481 does not participate in NAD binding. We hypothesize that the phenolic hydroxyl of Tyr-481 may be involved in the interaction of the toxin with substrate EF-2.     

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.     

Evidence for a two-base mechanism involving tyrosine-265 from arginine-219 mutants of alanine racemase

A positively charged residue, R219, was found to interact with the pyridine nitrogen of pyridoxal phosphate in the structure of alanine racemase from Bacillus stearothermophilus [Shaw et al. (1997) Biochemistry 36, 1329-1342]. Three site-directed mutants, R219K, R219A, and R219E, have been characterized and compared to the wild type enzyme (WT) to investigate the role of R219 in catalysis. The R219K mutation is functionally conservative, retaining approximately 25% of the WT activity. The R219A and R219E mutations decrease enzyme activity by approximately 100- and 1000-fold, respectively. These results demonstrate that a positively charged residue at this position is required for efficient catalysis. R219 and Y265 are connected through H166 via hydrogen bonds. The R219 mutants exhibit similar kinetic isotope effect trends: increased primary isotope effects (1.5-2-fold) but unchanged solvent isotope effects in the L --> D direction and increased solvent isotope effects (1.5-2-fold) but unchanged primary isotope effects in the D --> L direction. These results support a two-base racemization mechanism involving Y265 and K39. They additionally suggest that Y265 is selectively perturbed by R219 mutations through the H166 hydrogen-bond network. pH profiles show a large pKa shift from 7.1-7.4 (WT and R219K) to 9. 5-10.4 (R219A and R219E) for kcat/KM, and from 7.3 to 9.9-10.4 for kcat. The group responsible for this ionization is likely to be the phenolic hydroxyl of Y265, whose pKa is electrostatically perturbed in the WT by the H166-mediated interaction with R219. Accumulation of an absorbance band at 510 nm, indicative of a quinonoid intermediate, only in the D --> L direction with R219E provides additional evidence for a two-base mechanism involving Y265.     

"Catch 222," the effects of symmetry on ligand binding and catalysis in R67 dihydrofolate reductase as determined by mutations at Tyr-69

R67 dihydrofolate reductase (R67 DHFR) catalyzes the transfer of a hydride ion from NADPH to dihydrofolate, generating tetrahydrofolate. The homotetrameric enzyme provides a unique environment for catalysis as both ligands bind within a single active site pore possessing 222 symmetry. Mutation of one active site residue results in concurrent mutation of three additional symmetry-related residues, causing large effects on binding of both ligands as well as catalysis. For example, mutation of symmetry-related tyrosine 69 residues to phenylalanine (Y69F), results in large increases in Km values for both ligands and a 2-fold rise in the kcat value for the reaction (Strader, M. B., Smiley, R. D., Stinnett, L. G., VerBerkmoes, N. C., and Howell, E. E. (2001) Biochemistry 40, 11344-11352). To understand the interactions between specific Tyr-69 residues and each ligand, asymmetric Y69F mutants were generated that contain one to four Y69F mutations. A general trend observed from isothermal titration calorimetry and steady-state kinetic studies of these asymmetric mutants is that increasing the number of Y69F mutations results in an increase in the Kd and Km values. In addition, a comparison of steady-state kinetic values suggests that two Tyr-69 residues in one half of the active site pore are necessary for NADPH to exhibit a wild-type Km value. A tyrosine 69 to leucine mutant was also generated to approach the type(s) of interaction(s) occurring between Tyr-69 residues and the ligands. These studies suggest that the hydroxyl group of Tyr-69 is important for interactions with NADPH, whereas both the hydroxyl group and hydrophobic ring atoms of the Tyr-69 residues are necessary for proper interactions with dihydrofolate.     

Combined effects of two mutations of catalytic residues on the ketosteroid isomerase reaction

delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) catalyzes the isomerization of delta 5-3-ketosteroids to delta 4-3-ketosteroids by a conservative tautomeric transfer of the 4 beta-proton to the 6 beta-position with Tyr-14 as a general acid and Asp-38 as a general base [Kuliopulos, A., Mildvan, A. S., Shortle, D., & Talalay, P. (1989) Biochemistry 28, 149-159]. Primary, secondary, and combined deuterium kinetic isotope effects establish concerted substrate enolization to be the rate-limiting step with the wild-type enzyme [Xue, L., Talalay, P., & Mildvan, A. S. (1990) Biochemistry 29, 7491-7500]. The product of the fractional kcat values resulting from the Y14F mutation (10(-4.7)) and the D38N mutation (10(-5.6)) is comparable (10(-10.3)) to that of the double mutant Y14F + D38N (less than or equal to 10(-10.4)) which is completely inactive. Hence, the combined effects are either additive or synergistic. Quantitatively, similar effects of the two mutations on kcat/KM are found in the double mutant. Despite its inactivity, the Y14F + D38N double mutant forms crystals indistinguishable in form from those of the wild-type enzyme, tightly binds steroid substrates and substrate analogues, and immobilizes a spin-labeled steroid in an orientation indistinguishable from that found in the wild-type enzyme, indicating that the double mutant is otherwise largely intact. It is concluded that the total enzymatic activity of ketosteroid isomerase probably results from the independent and concerted functioning of Tyr-14 and Asp-38 in the rate-limiting enolization step, in accord with the perpendicular or antarafacial orientation of these two residues with respect to the enzyme-bound substrate. Synergistic effects of mutating two residues on kcat and on kcat/KM of enzyme-catalyzed multistep reactions are shown, theoretically, to occur when both residues act independently in the same step, and simple additivity occurs when this step is rate-limiting. Other conditions for additivity of the effects of mutations of kcat and kcat/KM are theoretically explored.     

Kinetic and ultraviolet spectroscopic studies of active-site mutants of delta 5-3-ketosteroid isomerase

delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) of Pseudomonas testosteroni promotes the highly efficient isomerization of delta 5-3-ketosteroids to delta 4-3-ketosteroids by means of a direct and stereospecific transfer of the 4 beta-proton to the 6 beta-position, via an enolic intermediate. An acidic residue responsible for the protonation of the 3-carbonyl function of the steroid and a basic group concerned with the proton transfer have been implicated in the catalytic mechanism. Recent NMR studies with a nitroxide spin-labeled substrate analogue have allowed positioning of the steroid into the 2.5-A X-ray crystal structure of the enzyme [Kuliopulos, A., Westbrook, E.M., Talalay, P., & Mildvan, A.S. (1987) Biochemistry 26, 3927-3937], thereby corroborating the approximate location of the steroid binding site deduced from a difference Fourier X-ray diffraction map of the 4-(acetoxymercuri)estradiol-isomerase complex [Westbrook, E.M., Piro, O.E., & Sigler, P.B. (1984) J. Biol. Chem. 259, 9096-9103]. The steroid lies in a hydrophobic cavity near Asp-38, Tyr-14, and Tyr-55. In order to assess the role of these amino acid residues in catalysis, the gene for isomerase was cloned, sequenced, and overexpressed in Escherichia coli [Kuliopulos, A., Shortle, D., & Talalay, P. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8893-8897], and the following mutants were prepared: Asp-38 to asparagine (D38N) and Tyr-14 and Tyr-55 to phenylalanine (Y14F and Y55F, respectively). The kcat value of the D38N mutant enzyme is 10(5.6)-fold lower than that of the wild-type enzyme, suggesting that Asp-38 functions as the base which abstracts the 4 beta-proton of the steroid in the rate-limiting step. Threefold lower Km values in all mutants indicate tighter binding of the substrate to the more hydrophobic sites. In comparison with the wild-type enzyme, the Y55F mutant shows only a 4-fold decrease in kcat while the Y14F mutant shows a 10(4.7)-fold decrease in kcat, suggesting that Tyr-14 is the general acid. The red shift of the ultraviolet absorption maximum of the competitive inhibitor 19-nortestosterone from 248 to 258-260 nm, which occurs upon binding to the wild-type enzyme [Wang, S.F., Kawahara, F.S., & Talalay, P. (1963) J. Biol. Chem. 238, 576-585], is mimicked in strong acid. This spectral shift was also observed with the D38N and Y55F mutants, but not on binding of the steroid to the Y14F mutant.(ABSTRACT TRUNCATED AT 400 WORDS)     

Asp-99 donates a hydrogen bond not to Tyr-14 but to the steroid directly in the catalytic mechanism of Delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B

Delta 5-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of Delta 5-3-ketosteroids at a rate approaching the diffusion limit by an intramolecular transfer of a proton. Despite the extensive studies on the catalytic mechanism, it still remains controversial whether the catalytic residue Asp-99 donates a hydrogen bond to the steroid or to Tyr-14. To clarify the role of Asp-99 in the catalysis, two single mutants of D99E and D99L and three double mutants of Y14F/D99E, Y14F/D99N, and Y14F/D99L have been prepared by site-directed mutagenesis. The D99E mutant whose side chain at position 99 is longer by an additional methylene group exhibits nearly the same kcat as the wild-type while the D99L mutant exhibits ca. 125-fold lower kcat than that of the wild-type. The mutations made at positions 14 and 99 exert synergistic or partially additive effect on kcat in the double mutants, which is inconsistent with the mechanism based on the hydrogen-bonded catalytic dyad, Asp-99 COOH...Tyr-14 OH...C3-O of the steroid. The crystal structure of D99E/D38N complexed with equilenin, an intermediate analogue, at 1.9 A resolution reveals that the distance between Tyr-14 O eta and Glu-99 O epsilon is ca. 4.2 A, which is beyond the range for a hydrogen bond, and that the distance between Glu-99 O epsilon and C3-O of the steroid is maintained to be ca. 2.4 A, short enough for a hydrogen bond to be formed. Taken together, these results strongly support the idea that Asp-99 contributes to the catalysis by donating a hydrogen bond directly to the intermediate.     

Studies of the mechanism of the delta 5-3-ketosteroid isomerase reaction by substrate, solvent, and combined kinetic deuterium isotope effects on wild-type and mutant enzymes

delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) catalyzes the isomerization of delta 5-3-ketosteroids to delta 4-3-ketosteroids by a conservative tautomeric transfer of the 4 beta-proton to the 6 beta-position using Tyr-14 as a general acid and Asp-38 as a general base [Kuliopulos, A., Mildvan, A. S., Shortle, D., & Talalay, P. (1989) Biochemistry 28, 149]. On deuteration of the 4 beta-position (97.0%) of the substrate, kcat(H)/kcat(4 beta-D) is 6.1 in H2O and 6.3 in D2O. The solvent isotope effect, kcat(H2O)/kcat(D2O), is 1.6 for both the 4 beta-H and 4 beta-D substrates. Mutation of Tyr-55 to Phe lowers kcat 4.3-fold; kcat(H)/kcat/4 beta-D) is 5.3 in H2O and 5.9 in D2O, and kcat(H2O)/kcat(D2O) with the 4 beta-H and 4 beta-D substrates is 1.5 and 1.7, respectively, indicating concerted general acid-base catalysis in either the enolization or the ketonization step of both the wild-type and the Tyr-55----Phe (Y55F) mutant enzymes. An additional slow step occurs with the Y55F mutant. Smaller isotope effects on Km are used to estimate individual rate constants in the kinetic schemes of both enzymes. On deuteration of the 4 alpha-position (88.6%) of the substrate, the secondary isotope effect on kcat/Km corrected for composition is 1.11 +/- 0.02 with the wild-type enzyme and 1.12 +/- 0.02 with the Y55F mutant. These effects decrease to 1.06 +/- 0.01 and 1.07 +/- 0.01, respectively, when the 4 beta-position is also deuterated, thereby establishing these to be kinetic (rather than equilibrium) secondary isotope effects and to involve a proton-tunneling contribution. Deuteration of the 6-position of the substrate (92.0%) produces no kinetic isotope effects on kcat/Km with either the wild-type (1.00 +/- 0.01) or the Y55F mutant (1.01 +/- 0.01) enzyme. Since a change in hybridization from sp3 to sp2 occurs at C-4 only during enolization of the substrate and a change in hybridization at C-6 from sp2 to sp3 occurs only during reketonization of the dienol intermediate, enolization of the substrate constitutes the concerted rate-limiting step. Concerted enolization is consistent with the right angle or antarafacial orientations of Tyr-14 and Asp-38 with respect to the enzyme-bound substrate and with the additive effects on kcat of mutation of these catalytic residues [Kuliopulos, A., Talalay, P., & Mildvan, A. S. (1990) Biophys. J. 57, 39a].     

Roles of aromatic residues in high interfacial activity of Naja naja atra phospholipase A2

Acidic phospholipase A2 (PLA2) from the venom of Chinese cobra (Naja naja atra) has high activity on zwitterionic membranes and contains six aromatic residues, including Tyr-3, Trp-18, Trp-19, Trp-61, Phe-64, and Tyr-110, on its putative interfacial binding surface. To assess the roles of these aromatic residues in the interfacial catalysis of N. n. atra PLA2, we mutated them to Ala and measured the effects on its interfacial catalysis. Enzymatic activities of the mutants toward various vesicle substrates and human neutrophils indicate that all but Trp-18 make significant contributions to interfacial catalysis. Among these aromatic residues, Trp-19, Trp-61, and Phe-64 play the most important roles. Binding affinities of the mutants for phospholipid-coated beads and their monolayer penetration indicate that Trp-19, Trp-61, and Phe-64 are critically involved in interfacial binding of N. n. atra PLA2 and penetrate into the membrane during the interfacial catalysis of N. n. atra PLA2. Further thermodynamic analysis suggests that the side chain of Phe-64 is fully inserted into the hydrophobic core of membrane whereas those of Trp-19 and Trp-61 are located in the membrane-water interface. Together, these results show that all three types of aromatic residues can play important roles in interfacial binding of PLA2 depending on their location and side-chain orientation. They also indicate that these aromatic side chains interact with membranes in distinct modes because of their different intrinsic preference for different parts of membranes.     

Beta-galactosidases (Escherichia coli) with double substitutions show that Tyr-503 acts independently of Glu-461 but cooperatively with Glu-537

Beta-galactosidases with single substitutions for Tyr-503, Glu-461, and Glu-537 and with double substitutions for Tyr-503 and either Glu-461 or Glu-537 were constructed. Control experiments showed that the very low kcat values obtained for the double-substituted enzymes were not a result of contamination, reversion, or nonactive site activity catalyzed on the surface of the proteins. Circular dichroism studies showed that the structures of the enzymes were intact. E461Q/Y503F-beta-galactosidase was inactivated in an "additive" manner. This indicated that Glu-461 and Tyr-503 act independently in catalysis. Because these residues are at opposite sides of the active site and act in different steps, this is expected. E537D/Y503F-beta-galactosidase was only inactivated a few-fold more than the most inactive of its two single-substituted constituent beta-galactosidases. This showed that Glu-537 and Tyr-503 interact cooperatively on the same step. This correlates well with the proposed role of Tyr-503 as an acid catalyst for the breakage of the covalent bond between Glu-537 and galactose.     

Redesigning the PheA domain of gramicidin synthetase leads to a new understanding of the enzyme's mechanism and selectivity

The PheA domain of gramicidin synthetase A, a non-ribosomal peptide synthetase, selectively binds phenylalanine along with ATP and Mg2+ and catalyzes the formation of an aminoacyl adenylate. In this study, we have used a novel protein redesign algorithm, K*, to predict mutations in PheA that should exhibit improved binding for tyrosine. Interestingly, the introduction of two predicted mutations to PheA did not significantly improve KD, as measured by equilibrium fluorescence quenching. However, the mutations improved the specificity of the enzyme for tyrosine (as measured by kcat/KM), primarily driven by a 56-fold improvement in KM, although the improvement did not make tyrosine the preferred substrate over phenylalanine. Using stopped-flow fluorometry, we examined binding of different amino acid substrates to the wild-type and mutant enzymes in the pre-steady state in order to understand the improvement in KM. Through these investigations, it became evident that substrate binding to the wild-type enzyme is more complex than previously described. These experiments show that the wild-type enzyme binds phenylalanine in a kinetically selective manner; no other amino acids tested appeared to bind the enzyme in the early time frame examined (500 ms). Furthermore, experiments with PheA, phenylalanine, and ATP reveal a two-step binding process, suggesting that the PheA-ATP-phenylalanine complex may undergo a conformational change toward a catalytically relevant intermediate on the pathway to adenylation; experiments with PheA, phenylalanine, and other nucleotides exhibit only a one-step binding process. The improvement in KM for the mutant enzyme toward tyrosine, as predicted by K*, may indicate that redesigning the side-chain binding pocket allows the substrate backbone to adopt productive conformations for catalysis but that further improvements may be afforded by modeling an enzyme:ATP:substrate complex, which is capable of undergoing conformational change.