Contribution of a Low-Barrier Hydrogen Bond to Catalysis Is Not Significant in Ketosteroid Isomerase

Low-barrier hydrogen bonds (LBHBs) have been proposed to have important influences on the enormous reaction rate increases achieved by many enzymes. Δ⁵-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of Δ⁵-3-ketosteroid to its conjugated Δ⁴-isomers at a rate that approaches the diffusion limit. Tyr14, a catalytic residue of KSI, has been hypothesized to form an LBHB with the oxyanion of a dienolate steroid intermediate generated during the catalysis. The unusual chemical shift of a proton at 16.8 ppm in the nuclear magnetic resonance spectrum has been attributed to an LBHB between Tyr14 Oη and C3-O of equilenin, an intermediate analogue, in the active site of D38N KSI. This shift in the spectrum was not observed in Y30F/Y55F/D38N and Y30F/Y55F/Y115F/D38N mutant KSIs when each mutant was complexed with equilenin, suggesting that Tyr14 could not form LBHB with the intermediate analogue in these mutant KSIs. The crystal structure of Y30F/Y55F/Y115F/D38N-equilenin complex revealed that the distance between Tyr14 Oη and C3-O of the bound steroid was within a direct hydrogen bond. The conversion of LBHB to an ordinary hydrogen bond in the mutant KSI reduced the binding affinity for the steroid inhibitors by a factor of 8.1–11. In addition, the absence of LBHB reduced the catalytic activity by only a factor of 1.7–2. These results suggest that the amount of stabilization energy of the reaction intermediate provided by LBHB is small compared with that provided by an ordinary hydrogen bond in KSI.     

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.     

Contribution of the hydrogen-bond network involving a tyrosine triad in the active site to the structure and function of a highly proficient ketosteroid isomerase from Pseudomonas putida biotype B

Delta(5)-3-Ketosteroid isomerase from Pseudomonas putida biotype B is one of the most proficient enzymes catalyzing an allylic isomerization reaction at rates comparable to the diffusion limit. The hydrogen-bond network (Asp99... Wat504...Tyr14...Tyr55...Tyr30) which links the two catalytic residues, Tyr14 and Asp99, to Tyr30, Tyr55, and a water molecule in the highly apolar active site has been characterized in an effort to identify its roles in function and stability. The DeltaG(U)(H2O) determined from equilibrium unfolding experiments reveals that the elimination of the hydroxyl group of Tyr14 or Tyr55 or the replacement of Asp99 with leucine results in a loss of conformational stability of 3.5-4.4 kcal/mol, suggesting that the hydrogen bonds of Tyr14, Tyr55, and Asp99 contribute significantly to stability. While decreasing the stability by about 6.5-7.9 kcal/mol, the Y55F/D99L or Y30F/D99L double mutation also reduced activity significantly, exhibiting a synergistic effect on k(cat) relative to the respective single mutations. These results indicate that the hydrogen-bond network is important for both stability and function. Additionally, they suggest that Tyr14 cannot function efficiently alone without additional support from the hydrogen bonds of Tyr55 and Asp99. The crystal structure of Y55F as determined at 1.9 A resolution shows that Tyr14 OH undergoes an alteration in orientation to form a new hydrogen bond with Tyr30. This observation supports the role of Tyr55 OH in positioning Tyr14 properly to optimize the hydrogen bond between Tyr14 and C3-O of the steroid substrate. No significant structural changes were observed in the crystal structures of Y30F and Y30F/Y55F, which allowed us to estimate approximately the interaction energies mediated by the hydrogen bonds Tyr30...Tyr55 and Tyr14...Tyr55. Taken together, our results demonstrate that the hydrogen-bond network provides the structural support that is needed for the enzyme to maintain the active-site geometry optimized for both function and stability.     

Detection of large pKa perturbations of an inhibitor and a catalytic group at an enzyme active site, a mechanistic basis for catalytic power of many enzymes

Delta(5)-3-Ketosteroid isomerase catalyzes cleavage and formation of a C-H bond at a diffusion-controlled limit. By determining the crystal structures of the enzyme in complex with each of three different inhibitors and by nuclear magnetic resonance (NMR) spectroscopic investigation, we evidenced the ionization of a hydroxyl group (pK(a) approximately 16.5) of an inhibitor, which forms a low barrier hydrogen bond (LBHB) with a catalytic residue Tyr(14) (pK(a) approximately 11.5), and the protonation of the catalytic residue Asp(38) with pK(a) of approximately 4.5 at pH 6.7 in the interaction with a carboxylate group of an inhibitor. The perturbation of the pK(a) values in both cases arises from the formation of favorable interactions between inhibitors and catalytic residues. The results indicate that the pK(a) difference between catalytic residue and substrate can be significantly reduced in the active site environment as a result of the formation of energetically favorable interactions during the course of enzyme reactions. The reduction in the pK(a) difference should facilitate the abstraction of a proton and thereby eliminate a large fraction of activation energy in general acid/base enzyme reactions. The pK(a) perturbation provides a mechanistic ground for the fast reactivity of many enzymes and for the understanding of how some enzymes are able to extract a proton from a C-H group with a pK(a) value as high as approximately 30.     

A theoretical model of the catalytic mechanism of the Delta5-3-ketosteroid isomerase reaction

The present paper describes a theoretical approach to the catalytic reaction mechanism involved in the conversion of 5-androstene-3,17-dione to 4-androstene-3,17-dione. The model incorporates the side chains of the residues tyrosine (Tyr(14)), aspartate (Asp(38)) and aspartic acid (Asp(99)) of the enzyme Delta(5)-3-ketosteroid isomerase (KSI; EC 5.3.3.1). The reaction involves two steps: first, Asp(38) acts as a base, abstracting the 4beta-H atom (proton) from C-4 of the steroid to form a dienolate as the intermediate; next, the intermediate is reketonized by proton transfer to the 6beta-position. Each step goes through its own transition state. Functional groups of the Tyr(14) and Asp(99) side chains act as hydrogen bond donors to the O1 atom of the steroid, providing stability along the reaction coordinate. Calculations were assessed at high level Hartree-Fock theory, using the 6-31G(*) basis set and the most important physicochemical properties involved in each step of the reaction, such as total energy, hardness, and dipole moment. Likewise, to explain the mechanism of reaction, highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), atomic orbital contributions to frontier orbitals formation, encoded electrostatic potentials, and atomic charges were used. Energy minima and transition state geometries were confirmed by vibrational frequency analysis. The mechanism described herein accounts for all of the properties, as well as the flow of atomic charges, explaining both catalytic mechanism and proficiency of KSI.     

Structure and enzymology of Delta5-3-ketosteroid isomerase.

The three-dimensional structures of Delta5-3-ketosteroid isomerases from two different bacterial species have been determined. The structures reveal an unusually apolar active site, in which each of several competitive inhibitors of the enzyme are held by two hydrogen bonds with the general acids Tyr14 and Asp99, and by hydrophobic interactions. The hydrogen bond between the Tyr14 hydroxyl and the C3 oxyanion of a transition-state analog is a low-barrier hydrogen bond, as indicated by a highly deshielded nuclear magnetic resonance. Structural and other biochemical studies have enabled the proposal of a detailed catalytic mechanism for Delta5-3-ketosteroid isomerase and provided a major thrust towards understanding the mechanism not only in chemical terms but also in energetics terms.     

Structure and enzymology of Δ-3-ketosteroid isomerase

The three-dimensional structures of Δ⁵-3-ketosteroid isomerases from two different bacterial species have been determined. The structures reveal an unusually apolar active site, in which each of several competitive inhibitors of the enzyme are held by two hydrogen bonds with the general acids Tyr14 and Asp99, and by hydrophobic interactions. The hydrogen bond between the Tyr14 hydroxyl and the C3 oxyanion of a transition-state analog is a low-barrier hydrogen bond, as indicated by a highly deshielded nuclear magnetic resonance. Structural and other biochemical studies have enabled the proposal of a detailed catalytic mechanism for Δ⁵-3-ketosteroid isomerase and provided a major thrust towards understanding the mechanism not only in chemical terms but also in energetics terms.     

NMR study of human mutant hemoglobins synthesized in Escherichia coli. Consequences of tyrosine alpha 42 substitutions

The hydroxyl group of Tyr alpha 42 in human hemoglobin forms a hydrogen bond with the carboxylate of Asp beta 99 which is considered to be one of the most important hydrogen bonds for stabilizing the "T-state." However, no spontaneous mutation at position 42 of the alpha subunit has been reported, and the role of the tyrosine has not been tested experimentally. Two artificial human mutant hemoglobins in which Tyr alpha 42 was replaced by phenylalanine or histidine were synthesized in Escherichia coli, and their proton NMR spectra were studied with particular attention to the hyperfine-shifted and hydrogen-bonded proton resonances. The site-directed mutagenesis of the Tyr alpha 42----Phe removes the hydrogen bond described above and prevents transition to the T-state so that the mutant Hb is rather similar to the "R-state" even when deoxygenated. On the other hand, the mutation from tyrosine to histidine causes less drastic structural changes, and its quaternary and tertiary structures are almost the same as native deoxy-Hb A. This may be attributed to the formation of a new hydrogen bond between His alpha 1(42) and Asp beta 2(99). These observations indicate that the hydrogen bond formed between Tyr alpha 42 and Asp beta 99 is required to convert unliganded Hb to the T-state.     

Hydrogen bonding in the active site of ketosteroid isomerase: electronic inductive effects and hydrogen bond coupling

Computational studies are performed to analyze the physical properties of hydrogen bonds donated by Tyr16 and Asp103 to a series of substituted phenolate inhibitors bound in the active site of ketosteroid isomerase (KSI). As the solution pK(a) of the phenolate increases, these hydrogen bond distances decrease, the associated nuclear magnetic resonance (NMR) chemical shifts increase, and the fraction of protonated inhibitor increases, in agreement with prior experiments. The quantum mechanical/molecular mechanical calculations provide insight into the electronic inductive effects along the hydrogen bonding network that includes Tyr16, Tyr57, and Tyr32, as well as insight into hydrogen bond coupling in the active site. The calculations predict that the most-downfield NMR chemical shift observed experimentally corresponds to the Tyr16-phenolate hydrogen bond and that Tyr16 is the proton donor when a bound naphtholate inhibitor is observed to be protonated in electronic absorption experiments. According to these calculations, the electronic inductive effects along the hydrogen bonding network of tyrosines cause the Tyr16 hydroxyl to be more acidic than the Asp103 carboxylic acid moiety, which is immersed in a relatively nonpolar environment. When one of the distal tyrosine residues in the network is mutated to phenylalanine, thereby diminishing this inductive effect, the Tyr16-phenolate hydrogen bond becomes longer and the Asp103-phenolate hydrogen bond shorter, as observed in NMR experiments. Furthermore, the calculations suggest that the differences in the experimental NMR data and electronic absorption spectra for pKSI and tKSI, two homologous bacterial forms of the enzyme, are due predominantly to the third tyrosine that is present in the hydrogen bonding network of pKSI but not tKSI. These studies also provide experimentally testable predictions about the impact of mutating the distal tyrosine residues in this hydrogen bonding network on the NMR chemical shifts and electronic absorption spectra.     

Catalytic activity of the D38A mutant of 3-oxo-Delta 5-steroid isomerase: recruitment of aspartate-99 as the base

3-oxo-Delta(5)-steroid isomerase (KSI) from Comamonas (Pseudomonas) testosteroni catalyzes the isomerization of beta,gamma-unsaturated 3-oxosteroids to their conjugated isomers through an intermediate dienolate. Residue Asp-38 (pK(a) 4.57) acts as a base to abstract a proton from C-4 of the substrate to form an intermediate dienolate, which is then reprotonated on C-6. Both Tyr-14 (pK(a) 11.6) and Asp-99 (pK(a) >/= 9.5) function as hydrogen-bond donors to O-3 of the steroid, helping to stabilize the transition states. Mutation of the active-site base Asp-38 to the weakly basic Asn (D38N) has previously been shown to result in a >10(8)-fold decrease of catalytic activity. In this work, we describe the preparation and kinetic analysis of the Ala-38 (D38A) mutant. Unexpectedly, D38A has a catalytic turnover number (k(cat)) that is ca. 10(6)-fold greater than the value for D38N and only about 140-fold less than that for wild type. Kinetic studies as a function of pH show that D38A-catalyzed isomerization involves two groups, with pK(a) values of 4.2 and 10.4, respectively, in the free enzyme, which are assigned to Asp-99 and either Tyr-14 or Tyr-55. A mechanism for D38A is proposed in which Asp-99 is recruited as the catalytic base, with stabilization of the intermediate dienolate ion and the flanking transition states provided by hydrogen bonding from both Tyr-14 and Tyr-55. This mechanism is supported by the lack of detectable activity of the D38A/D99N, D38A/Y14F, and D38A/Y55F double mutants.     

Direct observation by X-ray analysis of the tetrahedral "intermediate" of aspartic proteinases.

We report the X-ray analysis at 2.0 A resolution for crystals of the aspartic proteinase endothiapepsin (EC 3.4.23.6) complexed with a potent difluorostatone-containing tripeptide renin inhibitor (CP-81,282). The scissile bond surrogate, an electrophilic ketone, is hydrated in the complex. The pro-(R) (statine-like) hydroxyl of the tetrahedral carbonyl hydrate is hydrogen-bonded to both active-site aspartates 32 and 215 in the position occupied by a water in the native enzyme. The second hydroxyl oxygen of the hydrate is hydrogen-bonded only to the outer oxygen of Asp 32. These experimental data provide a basis for a model of the tetrahedral intermediate in aspartic proteinase-mediated cleavage of the amide bond. This indicates a mechanism in which Asp 32 is the proton donor and Asp 215 carboxylate polarizes a bound water for nucleophilic attack. The mechanism involves a carboxylate (Asp 32) that is stabilized by extensive hydrogen bonding, rather than an oxyanion derivative of the peptide as in serine proteinase catalysis.     

A low-barrier hydrogen bond between histidine of secreted phospholipase A2 and a transition state analog inhibitor

This work describes in-depth NMR characterization of a unique low-barrier hydrogen bond (LBHB) between an active site residue from the enzyme and a bound inhibitor: the complex between secreted phospholipase A(2) (sPLA(2), from bee venom and bovine pancreas) and a transition-state analog inhibitor HK32. A downfield proton NMR resonance, at 17-18 ppm, was observed in the complex but not in the free enzyme. On the basis of site-specific mutagenesis and specific 15N-decoupling, this downfield resonance was assigned to the active site H48, which is part of the catalytic dyad D99-H48. These results led to a hypothesis that the downfield resonance represents the proton (H(epsilon 2) of H48) involved in the H-bonding between D99 and H48, in analogy with serine proteases. However, this was shown not to be the case by use of the bovine enzyme labeled with specific [15N(epsilon 2)]His. Instead, the downfield resonance arises from H(delta1) of H48, which forms a hydrogen bond with a non-bridging phosphonate oxygen of the inhibitor. Further studies showed that this proton displays a fractionation factor of 0.62(+/-0.06), and an exchange rate protection factor of >100 at 285 K and >40 at 298 K, which are characteristic of a LBHB. The pK(a) of the imidazole ring of H48 was shown to be shifted from 5.7 for the free enzyme to an apparent value of 9.0 in the presence of the inhibitor. These properties are very similar to those of the Asp em leader His LBHBs in serine proteases. Possible structural bases and functional consequences for the different locations of the LBHB between these two types of enzymes are discussed. The results also underscore the importance of using specific isotope labeling, rather than extrapolation of NMR results from other enzyme systems, to assign the downfield proton resonance to a specific hydrogen bond. Although our studies did not permit the strength of the LBHB to be accurately measured, the data do not provide support for an unusually strong hydrogen bond strength (i.e. >10 kcal/mol).     

Electrostatic effects and binding determinants in the catalysis of prolyl oligopeptidase. Site specific mutagenesis at the oxyanion binding site

Prolyl oligopeptidase, a member of a new family of serine peptidases, plays an important role in memory disorders. Earlier x-ray crystallographic investigations indicated that stabilization of the tetrahedral transition state of the reaction involved hydrogen bond formation between the oxyanion of the tetrahedral intermediate and the OH group of Tyr(473). The contribution of the OH group was tested with the Y473F variant using various substrates. The charged succinyl-Gly-Pro-4-nitroanilide was hydrolyzed with a much lower k(cat)/K(m) compared with the neutral benzyloxycarbonyl-G1y-Pro-2-naphthylamide, although the binding modes of the two substrates were similar, as shown by x-ray crystallography. This suggested that electrostatic interactions between Arg(643) and the succinyl group competed with the productive binding mechanism. Unlike most enzyme reactions, catalysis by the wild-type enzyme exhibited positive activation entropy. In contrast, the activation entropy for the Y473F variant was negative, suggesting that the tyrosine OH group is involved in stabilizing both the transition state and the water shell at the active site. Importantly, Tyr(473) is also implicated in the formation of the enzyme-substrate complex. The nonlinear Arrhenius plot suggested a greater significance of the oxyanion binding site at physiological temperature. The results indicated that Tyr(473) was more needed at high pH, at high temperature, and with charged substrates exhibiting "internally competitive inhibition."     

Binding of 2-naphthols to D38E mutants of 3-oxo-Delta 5-steroid isomerase: variation of ligand ionization state with the nature of the electrophilic component

3-Oxo-Delta(5)-steroid isomerase (KSI) catalyzes the isomerization of a variety of 3-oxo-Delta(5)-steroids to their conjugated Delta(4) isomers. The mechanism involves sequential enolization and ketonization, with Asp-38 acting to transfer a proton from C-4 to C-6 through a dienol(ate) intermediate. We have previously proposed that this intermediate is anionic, with stabilization provided from direct hydrogen bonding from Tyr-14 and Asp-99 to the oxygen of the steroid. In this work, we analyze the binding of substituted 2-naphthols, which are analogues of the intermediate dienol, to the D38E KSI mutant and the corresponding double mutants lacking one of the two electrophilic groups (D38E/Y14F and D38E/D99A). The binding of these naphthols to the mutant KSIs at pH 7 is described by the modified Bronsted equation: log K(D) = alpha(pK(a)) + constant, where K(D) is the dissociation constant of the complex. The high value of alpha for D38E (alpha = 0.87 +/- 0.06) indicates that the negative charge in these D38E-naphthol complexes is localized almost exclusively on the bound ligand. In contrast, values of alpha for the double mutants (alpha = 0.28 +/- 0.02 for D38E/Y14F and alpha = 0.25 +/- 0.02 for D38E/D99A) are consistent with very little negative charge on the oxygen of the bound naphthol. Ultraviolet spectra of 5-nitro-2-naphthol and the fluorescence spectra of equilenin bound to these mutants support this interpretation. Extrapolation of these results to the intermediate in the catalytic reaction suggests that for the reaction with D38E, the intermediate is a negatively charged dienolate with hydrogen bonding from both Tyr-14 and Asp-99. Removal of either one of these H-bond donors (Tyr-14 or Asp-99) causes destabilization of the anion and results in a dienol enzyme-intermediate complex rather than a dienolate.     

The catalytic power of ketosteroid isomerase investigated by computer simulation

Ketosteroid isomerase (KSI) catalyzes the isomerization of Delta(5)-3-ketosteroids and Delta(4)-3-ketosteroids at very high rates. Here we examine the principles underlying the catalytic efficiency of KSI by computer simulations using the empirical valence bond method in combination with molecular dynamics free energy perturbation simulations. The simulations reproduce available kinetic and structural data very well and allow us to examine several features of the catalytic mechanism in detail. It is found that about 60% of the rate enhancement is due to stabilization of the negatively charged dienolate intermediate by hydrogen bonding. The critical H-bond between Tyr16 and the intermediate is found to be a normal ionic H-bond with the preferred proton location on the tyrosine residue. The remaining 40% of the catalytic effect originates from a reduction of the reorganization energy of the reaction. The possibility of an active site water molecule occupying the empty cavity adjacent to the catalytic base (Asp40) is also addressed. The existence of such a water molecule could explain how the enzyme manages to maintain a low pK(a) for the general base residue.     

Five atomic resolution structures of endothiapepsin inhibitor complexes: implications for the aspartic proteinase mechanism

Endothiapepsin is derived from the fungus Endothia parasitica and is a member of the aspartic proteinase class of enzymes. This class of enzyme is comprised of two structurally similar lobes, each lobe contributing an aspartic acid residue to form a catalytic dyad that acts to cleave the substrate peptide bond. The three-dimensional structures of endothiapepsin bound to five transition state analogue inhibitors (H189, H256, CP-80,794, PD-129,541 and PD-130,328) have been solved at atomic resolution allowing full anisotropic modelling of each complex. The active sites of the five structures have been studied with a view to studying the catalytic mechanism of the aspartic proteinases by locating the active site protons by carboxyl bond length differences and electron density analysis. In the CP-80,794 structure there is excellent electron density for the hydrogen on the inhibitory statine hydroxyl group which forms a hydrogen bond with the inner oxygen of Asp32. The location of this proton has implications for the catalytic mechanism of the aspartic proteinases as it is consistent with the proposed mechanism in which Asp32 is the negatively charged aspartate. A number of short hydrogen bonds (approximately 2.6 A) with ESD values of around 0.01 A that may have a role in catalysis have been identified within the active site of each structure; the lengths of these bonds have been confirmed using NMR techniques. The possibility and implications of low barrier hydrogen bonds in the active site are considered.     

Role of the oxyanion binding site and subsites S1 and S2 in the catalysis of oligopeptidase B, a novel target for antimicrobial chemotherapy

Oligopeptidase B is a member of a novel serine peptidase family, found in Gram-negative bacteria and trypanosomes. The enzyme is involved in host cell invasion, and thus, it is an important target for drug design. Oligopeptidase B is specific for substrates with a pair of basic residues at positions P1 and P2. The sensitivity of substrates to high ionic strength suggests that the arginines interact with the carboxylate ions of the enzyme. On the basis of a three-dimensional model, two carboxyl dyads (Asp460 and Asp462 and Glu576 and Glu578) can be assigned as binding sites for arginines P1 and P2, respectively. The dyads are involved in several events: (i) substrate binding, (ii) substrate inhibition at high substrate concentrations (different inhibitory mechanisms were demonstrated with substrates bearing one and two arginine residues), (iii) enzyme activation at millimolar CaCl2 concentrations with substrates having one arginine, and (iv) interaction of Ca2+ with the dyads which simplified the complex pH dependence curves. Titration with a product-like inhibitor revealed the pK(a) of the carboxyl group that perturbed the pH-kcat/Km profiles. The OH group of Tyr452 is part of the oxyanion binding site, which stabilizes the transition state of the reaction. Its role studied with the Tyr452Phe variant indicates that (i) the catalytic contribution of the OH group depends on the substrate and (ii) the catalysis is, unusually, an entropy-driven process at physiological temperature. The NH group of the scissile peptide bond accounts for the deviation of the reaction from the Eyring plot above 25 degrees C, and for abolishing potential nonproductive binding.     

The catalytic role of aspartate in a short strong hydrogen bond of the Asp274-His32 catalytic dyad in phosphatidylinositol-specific phospholipase C can be substituted by a chloride ion

Phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis catalyzes the cleavage of the phosphorus-oxygen bond in phosphatidylinositol. The focus of this work is to dissect the roles of the carboxylate side chain of Asp(274) in the Asp(274)-His(32) dyad, where a short strong hydrogen bond (SSHB) was shown to exist based on NMR criteria. A regular hydrogen bond (HB) was observed in D274N, and no low field proton resonance was detected for D274E and D274A. Comparison of the activity of wild type, D274N, and D274A suggested that the regular HB contributes significantly (approximately 4 kcal/mol) to catalysis, whereas the SSHB contributes only an additional 2 kcal/mol. The mutant D274E displays high activity similar to wild type, suggesting that the negative charge is sufficient for the catalytic role of Asp(274). To further support this interpretation and rule out possible contribution of regular HB or SSHB in D274E, we showed that the activity of D274G can be rescued by exogenous chloride ions to a level comparable with that of D274E. Comparison between different anions suggested that the ability of an anion to rescue the activity is due to the size and the charge of the anion not the property as a HB acceptor. In conclusion, a major fraction of the functional role of Asp(274) in the Asp(274)-His(32) dyad can be attributed to a negative charge (as in D274E and D274G-Cl(-)), and the SSHB in the wild type enzyme provides minimal contribution to catalysis. These results represent novel insight for an Asp-His catalytic dyad and for the mechanism of phosphatidylinositol-specific phospholipase C.     

The roles of Tyr(91) and Lys(162) in general acid-base catalysis in the pigeon NADP+-dependent malic enzyme

The role of general acid-base catalysis in the enzymatic mechanism of NADP+-dependent malic enzyme was examined by detailed steady-state kinetic studies through site-directed mutagenesis of the Tyr(91) and Lys(162) residues in the putative catalytic site of the enzyme. Y91F and K162A mutants showed approx. 200- and 27000-fold decreases in k(cat) values respectively, which could be partially recovered with ammonium chloride. Neither mutant had an effect on the partial dehydrogenase activity of the enzyme. However, both Y91F and K162A mutants caused decreases in the k(cat) values of the partial decarboxylase activity of the enzyme by approx. 14- and 3250-fold respectively. The pH-log(k(cat)) profile of K162A was found to be different from the bell-shaped profile pattern of wild-type enzyme as it lacked a basic pK(a) value. Oxaloacetate, in the presence of NADPH, can be converted by malic enzyme into L-malate by reduction and into enolpyruvate by decarboxylation activities. Compared with wild-type, the K162A mutant preferred oxaloacetate reduction to decarboxylation. These results are consistent with the function of Lys(162) as a general acid that protonates the C-3 of enolpyruvate to form pyruvate. The Tyr(91) residue could form a hydrogen bond with Lys(162) to act as a catalytic dyad that contributes a proton to complete the enol-keto tautomerization.     

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

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