Substrate gating confers steroid specificity to estrogen sulfotransferase.

Estrogen sulfotransferase (EST) exhibits a high substrate specificity and catalytic efficiency toward estrogens such as estradiol (E2) but insignificant ability to sulfate hydroxysteroids such as dehydroepiandrosterone (DHEA). To provide the structural basis for this estrogen specificity, we mutated amino acid residues that constitute the substrate-binding site of EST. Among these mutants, only Tyr-81 decreased E2 and increased DHEA sulfotransferase activities. Substitution for Tyr-81 by smaller hydrophobic residues increased K(m(E2)) for E2 activity, whereas the k(cat(E2)) remained relatively constant. The Y81L mutant exhibited the same DHEA activity as wild-type hydroxysteroid sulfotransferase, for which K(m(DHEA)) remained relatively constant, and k(cat(DHEA)) was markedly increased. The side chain of Tyr-81 is directed at the A-ring of the E2 molecule in the substrate-binding pocket of EST, constituting a steric gate with Phe-142 sandwiching E2 from the opposite side. The present mutagenesis study indicates that the 3beta-hydroxyl group of the DHEA molecule is excluded from the catalytic site of EST through steric hindrance of Tyr-81 with the C-19 methyl group of DHEA. Thus, this stricture-like gating caused by steric hindrance appears to be a structural principle for conferring estrogen specificity to EST.     

Alanine scanning mutagenesis of the testosterone binding site of rat 3 alpha-hydroxysteroid dehydrogenase demonstrates contact residues influence the rate-determining step

Aldo-keto reductase (AKR1C) isoforms can regulate ligand access to nuclear receptors by acting as hydroxysteroid dehydrogenases. The principles that govern steroid hormone binding and steroid turnover by these enzymes were analyzed using rat 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD, AKR1C9) as the protein model. Systematic alanine scanning mutagenesis was performed on the substrate-binding pocket as defined by the crystal structure of the 3alpha-HSD.NADP(+).testosterone ternary complex. T24, L54, F118, F129, T226, W227, N306, and Y310 were individually mutated to alanine, while catalytic residues Y55 and H117 were unaltered. The effects of these mutations on the ordered bi-bi mechanism were examined. No mutations changed the affinity for NADPH by more than 2-3-fold. Fluorescence titrations of the energy transfer band of the E.NADPH complex with competitive inhibitors testosterone and progesterone showed that the largest effect was a 23-fold decrease in the affinity for progesterone in the W227A mutant. By contrast, changes in the K(d) for testosterone were negligible. Examination of the k(cat)/K(m) data for these mutants indicated that, irrespective of steroid substrate, the bimolecular rate constant was more adversely affected when alanine replaced an aromatic hydrophobic residue. By far, the greatest effects were on k(cat) (decreases of more than 2 log units), suggesting that the rate-determining step was either altered or slowed significantly. Single- and multiple-turnover experiments for androsterone oxidation showed that while the wild-type enzyme demonstrated a k(lim) and burst kinetics consistent with slow product release, the W227A and F118A mutants eliminated this kinetic profile. Instead, single- and multiple-turnover experiments gave k(lim) and k(max) values identical with k(cat) values, respectively, indicating that chemistry was now rate-limiting overall. Thus, conserved residues within the steroid-binding pocket affect k(cat) more than K(d) by influencing the rate-determining step of steroid oxidation. These findings support the concept of enzyme catalysis in which the correct positioning of reactants is essential; otherwise, k(cat) will be limited by the chemical event.     

Removal of distal protein-water hydrogen bonds in a plant epoxide hydrolase increases catalytic turnover but decreases thermostability

A putative proton wire in potato soluble epoxide hydrolase 1, StEH1, was identified and investigated by means of site-directed mutagenesis, steady-state kinetic measurements, temperature inactivation studies, and X-ray crystallography. The chain of hydrogen bonds includes five water molecules coordinated through backbone carbonyl oxygens of Pro(186), Leu(266), His(269), and the His(153) imidazole. The hydroxyl of Tyr(149) is also an integrated component of the chain, which leads to the hydroxyl of Tyr(154). Available data suggest that Tyr(154) functions as a final proton donor to the anionic alkylenzyme intermediate formed during catalysis. To investigate the role of the putative proton wire, mutants Y149F, H153F, and Y149F/H153F were constructed and purified. The structure of the Y149F mutant was solved by molecular replacement and refined to 2.0 A resolution. Comparison with the structure of wild-type StEH1 revealed only subtle structural differences. The hydroxyl group lost as a result of the mutation was replaced by a water molecule, thus maintaining a functioning hydrogen bond network in the proton wire. All mutants showed decreased catalytic efficiencies with the R,R-enantiomer of trans-stilbene oxide, whereas with the S,S-enantiomer, k (cat)/K (M) was similar or slightly increased compared with the wild-type reactions. k (cat) for the Y149F mutant with either TSO enantiomer was increased; thus the lowered enzyme efficiencies were due to increases in K (M). Thermal inactivation studies revealed that the mutated enzymes were more sensitive to elevated temperatures than the wild-type enzyme. Hence, structural alterations affecting the hydrogen bond chain caused increases in k (cat) but lowered thermostability.     

Active-site residues governing high steroid isomerase activity in human glutathione transferase A3-3

Glutathione transferase (GST) A3-3 is the most efficient human steroid double-bond isomerase known. The activity with Delta(5)-androstene-3,17-dione is highly dependent on the phenolic hydroxyl group of Tyr-9 and the thiolate of glutathione. Removal of these groups caused an 1.1 x 10(5)-fold decrease in k(cat); the Y9F mutant displayed a 150-fold lower isomerase activity in the presence of glutathione and a further 740-fold lower activity in the absence of glutathione. The Y9F mutation in GST A3-3 did not markedly decrease the activity with the alternative substrate 1-chloro-2,4-dinitrobenzene. Residues Phe-10, Leu-111, and Ala-216 selectively govern the activity with the steroid substrate. Mutating residue 111 into phenylalanine caused a 25-fold decrease in k(cat)/K(m) for the steroid isomerization. The mutations A216S and F10S, separate or combined, affected the isomerase activity only marginally, but with the additional L111F mutation k(cat)/K(m) was reduced to 0.8% of that of the wild-type value. In contrast, the activities with 1-chloro-2,4-dinitrobenzene and phenethylisothiocyanate were not largely affected by the combined mutations F10S/L111F/A216S. K(i) values for Delta(5)-androstene-3,17-dione and Delta(4)-androstene-3,17-dione were increased by the triple mutation F10S/L111F/A216S. The pK(a) of the thiol group of active-site-bound glutathione, 6.1, increased to 6.5 in GST A3-3/Y9F. The pK(a) of the active-site Tyr-9 was 7.9 for the wild-type enzyme. The pH dependence of k(cat)/K(m) of wild-type GST A3-3 for the isomerase reaction displays two kinetic pK(a) values, 6.2 and 8.1. The basic limb of the pH dependence of k(cat) and k(cat)/K(m) disappears in the Y9F mutant. Therefore, the higher kinetic pK(a) reflects ionization of Tyr-9, and the lower one reflects ionization of glutathione. We propose a reaction mechanism for the double-bond isomerization involving abstraction of a proton from C4 in the steroid accompanied by protonation of C6, the thiolate of glutathione serving as a base and Tyr-9 assisting by polarizing the 3-oxo group of the substrate.     

Improved catalytic properties of halohydrin dehalogenase by modification of the halide-binding site

Halohydrin dehalogenase (HheC) from Agrobacterium radiobacter AD1 catalyzes the dehalogenation of vicinal haloalcohols by an intramolecular substitution reaction, resulting in the formation of the corresponding epoxide, a halide ion, and a proton. Halide release is rate-limiting during the catalytic cycle of the conversion of (R)-p-nitro-2-bromo-1-phenylethanol by the enzyme. The recent elucidation of the X-ray structure of HheC showed that hydrogen bonds between the OH group of Tyr187 and between the Odelta1 atom of Asn176 and Nepsilon1 atom of Trp249 could play a role in stabilizing the conformation of the halide-binding site. The possibility that these hydrogen bonds are important for halide binding and release was studied using site-directed mutagenesis. Steady-state kinetic studies revealed that mutant Y187F, which has lost both hydrogen bonds, has a higher catalytic activity (k(cat)) with two of the three tested substrates compared to the wild-type enzyme. Mutant W249F also shows an enhanced k(cat) value with these two substrates, as well as a remarkable increase in enantiopreference for (R)-p-nitro-2-bromo-1-phenylethanol. In case of a mutation at position 176 (N176A and N176D), a 1000-fold lower catalytic efficiency (k(cat)/K(m)) was obtained, which is mainly due to an increase of the K(m) value of the enzyme. Pre-steady-state kinetic studies showed that a burst of product formation precedes the steady state, indicating that halide release is still rate-limiting for mutants Y187F and W249F. Stopped-flow fluorescence experiments revealed that the rate of halide release is 5.6-fold higher for the Y187F mutant than for the wild-type enzyme and even higher for the W249F enzyme. Taken together, these results show that the disruption of two hydrogen bonds around the halide-binding site increases the rate of halide release and can enhance the overall catalytic activity of HheC.     

Membrane binding and substrate access merge in cytochrome P450 7A1, a key enzyme in degradation of cholesterol.

To study membrane topology and mechanism for substrate specificity, we truncated residues 2-24 in microsomal cytochrome P450 7A1 (P450 7A1) and introduced conservative and nonconservative substitutions at positions 214-227. Heterologous expression in Escherichia coli was followed by investigation of the subcellular distribution of the mutant P450s and determination of the kinetic and substrate binding parameters for cholesterol. The results indicate that a hydrophobic region, comprising residues 214-227, forms a secondary site of attachment to the membrane in P450 7A1 in addition to the NH(2)-terminal signal-anchor sequence. There are two groups of residues at this enzyme-membrane interface. The first are those whose mutation results in more cytosolic P450 (Val-214, His-225, and Met-226). The second group are those whose mutation leads to more membrane-bound P450 (Phe-215, Leu-218, Ile-224, and Phe-227). In addition, the V214A, V214L, V214T, F215A, F215L, F215Y, L218I, L218V, V219T, and M226A mutants showed a 5-12-fold increased K(m) for cholesterol. The k(cat) of the V214A, V214L, V219T, and M226A mutants was increased up to 1.8-fold, and that of the V214T, F215A, F215L, F215Y, L218I, and L218V mutants was decreased 3-10.5-fold. Based on analysis of these mutations we suggest that cholesterol enters P450 7A1 through the membrane, and Val-214, Phe-215, and Leu-218 are the residues located near the point of cholesterol entry. The results provide an understanding of both the P450 7A1-membrane interactions and the mechanism for substrate specificity.     

Amphipathic property of free thiol group contributes to an increase in the catalytic efficiency of carboxypeptidase Y.

Cys341 of carboxypeptidase Y, which constitutes one side of the solvent-accessible surface of the S1 binding pocket, was replaced with Gly, Ser, Asp, Val, Phe or His by site-directed mutagenesis. Kinetic analysis, using Cbz-dipeptide substrates, revealed that polar amino acids at the 341 position increased K(m) whereas hydrophobic amino acids in this position tended to decrease K(m). This suggests the involvement of Cys341 in the formation of the Michaelis complex in which Cys341 favors the formation of hydrophobic interactions with the P1 side chain of the substrate as well as with residues comprising the surface of the S1 binding pocket. Furthermore, C341G and C341S mutants had significantly higher k(cat) values with substrates containing the hydrophobic P1 side chain than C341V or C341F. This indicates that the nonhydrophobic property conferred by Gly or Ser gives flexibility or instability to the S1 pocket, which contributes to the increased k(cat) values of C341G or C341S. The results suggest that Cys341 may interact with His397 during catalysis. Therefore, we propose a dual role for Cys341: (a) its hydrophobicity allows it to participate in the formation of the Michaelis complex with hydrophobic substrates, where it maintains an unfavorable steric constraint in the S1 subsite; (b) its interaction with the imidazole ring of His397 contributes to the rate enhancement by stabilizing the tetrahedral intermediate in the transition state.     

The structural basis for the perturbed pKa of the catalytic base in 4-oxalocrotonate tautomerase: kinetic and structural effects of mutations of Phe-50

The amino-terminal proline of 4-oxalocrotonate tautomerase (4-OT) functions as the general base catalyst in the enzyme-catalyzed isomerization of beta,gamma-unsaturated enones to their alpha,beta-isomers because of its unusually low pK(a) of 6.4 +/- 0.2, which is 3 units lower than that of the model compound, proline amide. Recent studies show that this abnormally low pK(a) is not due to the electrostatic effects of nearby cationic residues (Arg-11, Arg-39, and Arg-61) [Czerwinski, R. M., Harris, T. K., Johnson, Jr., W. H., Legler, P. M., Stivers, J. T., Mildvan, A. S., and Whitman, C. P. (1999) Biochemistry 38, 12358-12366]. Hence, it may result solely from a low local dielectric constant of 14.7 +/- 0.8 at the otherwise hydrophobic active site. Support for this mechanism comes from the study of mutants of the active site Phe-50, which is 5.8 A from Pro-1 and is one of 12 apolar residues within 9 A of Pro-1. Replacing Phe-50 with Tyr does not significantly alter k(cat) or K(m) and results in a pK(a) of 6.0 +/- 0.1 for Pro-1 as determined by (15)N NMR spectroscopy, comparable to that observed for wild type. (1)H-(15)N HSQC and 3D (1)H-(15)N NOESY HSQC spectra of the F50Y mutant demonstrate its conformation to be very similar to that of the wild-type enzyme. In the F50Y mutant, the pK(a) of Tyr-50 is increased by two units from that of a model compound N-acetyl-tyrosine amide to 12.2 +/- 0.3, as determined by UV and (1)H NMR titrations, yielding a local dielectric constant of 13.4 +/- 1.7, in agreement with the value of 13.7 +/- 0.3 determined from the decreased pK(a) of Pro-1 in this mutant. In the F50A mutant, the pK(a) of Pro-1 is 7.3 +/- 0.1 by (15)N NMR titration, comparable to the pK(a) of 7.6 +/- 0.2 found in the pH vs k(cat)/K(m) rate profile, and is one unit greater than that of the wild-type enzyme, indicating an increase in the local dielectric constant to a value of 21.2 +/- 2.6. A loss of structure of the beta-hairpin from residues 50 to 57, which covers the active site, and is the site of the mutation, is indicated by the disappearance in the F50A mutant of four interstrand NOEs and one turn NOE found in wild-type 4-OT. (1)H-(15)N HSQC spectra of the F50A mutant reveal widespread and large changes in the backbone (15)N and NH chemical shifts including those of Gly residues 48, 51, 53, and 54 causing their loss of dispersion at 23 degrees C and their disappearance at 43 degrees C due to rapid exchange with solvent. These observations confirm that the active site of the F50A mutant is more accessible to the external aqueous environment, causing an increase in the local dielectric constant and in the pK(a) of Pro-1. In addition, the F50A mutation decreased k(cat) 167-fold and increased K(m) 11-fold from those of the wild-type enzyme, suggesting an important role for the hydrophobic environment in catalysis, beyond that of decreasing the pK(a) of Pro-1. The F50I and F50V mutations destabilize the protein and decrease k(cat) by factors of 58 and 1.6, and increase K(m) by 3.3- and 3.8-fold, respectively.     

Structural determinants of Cys2His2 zinc fingers.

Two mutants of the zinc finger peptide Xfin-31 (Ac-YKCGLCERSFVEKSALSRHQRVHKN-CONH2) containing alterations to the conserved hydrophobic core have been constructed and their zinc-bound structures investigated by 1H NMR techniques. In the first (Xfin-31B) a double mutation R8F/F10G places the conserved core aromatic residue at position 8 rather than position 10. In the second (Xfin-31C), Phe-10 is replaced by Leu. A qualitative analysis of 1H chemical shifts, NOE connectivities and coupling constants indicates that the global folds of both mutants are similar to that of the wild-type protein. However, amide exchange rates suggest that the F10L mutant is much less stable than either the wild-type or the R8F/F10G mutant.     

Analysis of a conserved hydrophobic pocket important for the thermostability of Bacillus pumilus chloramphenicol acetyltransferase (CAT-86)

Site-directed mutagenesis was carried out on Bacillus pumilus chloramphenicol acetyltransferase (CAT-86) to determine the effects of substitution at a conserved hydrophobic pocket identified earlier as important for thermostability. Mutations were introduced that would substitute residues at consensus positions 33, 191 and 203 in the enzyme, both individually and in combination. Two mutants, SDM1 (CAT-86 Y33F, A203V) and SDM5 (CAT-86 A203I), were more thermostable than wild-type and two mutants, SDM4 (CAT-86 I191V) and SDM7 (CAT-86 A203G), were less stable. Reconstruction of the residues of this hydrophobic pocket to that of a more thermostable CAT-R387 enzyme pocket (as a Y33F, I191V, A203V triple mutant) increased the thermostability of the enzyme above the wild-type, but its stability was less than that of SDM1 and SDM5. The K(m) values of the mutant enzymes for chloramphenicol and acetyl-CoA were essentially unaltered (in the ranges 15-30 and 26-35 microM respectively) and the specific activity of purified enzyme was in the range 270-710 units/mg protein. The possible effects of the amino acid substitutions on the CAT-86 structure were determined by homology modelling. A reduction in conformational strain and optimized hydrophobic interactions are predicted to be responsible for the increased thermostability of the SDM1 and SDM5 mutants.     

Roles of active site aromatic residues in catalysis by ketosteroid isomerase from Pseudomonas putida biotype B.

The aromatic residues Phe-54, Phe-82, and Trp-116 in the hydrophobic substrate-binding pocket of Delta(5)-3-ketosteroid isomerase from Pseudomonas putida biotype B have been characterized in their roles in steroid binding and catalysis. Kinetic and equilibrium binding analyses were carried out for the mutant enzymes with the substitutions Phe-54 --> Ala or Leu, Phe-82 --> Ala or Leu, and Trp-116 --> Ala, Phe, or Tyr. The removal of their bulky, aromatic side chains at any of these three positions results in reduced k(cat), particularly when Phe-82 or Trp-116 is replaced by Ala. The results are consistent with the binding interactions of the aromatic residues with the bound steroid contributing to catalysis. All the mutations except the F82A mutation increase K(m); the F82A mutation decreases K(m) by ca. 3-fold, suggesting a possibility that the phenyl ring at position 82 might be unfavorable for substrate binding. The K(D) values for d-equilenin, an intermediate analogue, suggest that a space-filling hydrophobic side chain at position 54, a phenyl ring at position 82, and a nonpolar aromatic or small side chain at position 116 might be favorable for binding the reaction intermediate. In contrast to the increased K(D) for equilenin, the enzymes with any substitutions at positions 54 and 116 display a decreased K(D) for 19-nortestosterone, a product analogue, indicating that Phe-54 and Trp-116 might be unfavorable for product binding. The crystal structure of F82A determined to 2.1-A resolution reveals that Phe-82 is important for maintaining the active site geometry. Taken together, our results demonstrate that Phe-54, Phe-82, and Trp-116 contribute differentially to the stabilization of steroid species including substrate, intermediate, and product.     

Manipulation of the active site loops of D-hydantoinase, a (beta/alpha)8-barrel protein, for modulation of the substrate specificity

We previously proposed that the stereochemistry gate loops (SGLs) constituting the substrate binding pocket of D-hydantoinase, a (beta/alpha)(8)-barrel enzyme, might be major structural determinants of the substrate specificity [Cheon, Y. H., et al. (2002) Biochemistry 41, 9410-9417]. To construct a mutant D-hydantoinase with favorable substrate specificity for the synthesis of commercially important non-natural amino acids, the SGL loops of the enzyme were rationally manipulated on the basis of the structural analysis and sequence alignment of three hydantoinases with distinct substrate specificities. In the SGLs of D-hydantoinase from Bacillus stearothermophilus SD1, mutations of hydrophobic and bulky residues Met 63, Leu 65, Phe 152, and Phe 159, which interact with the exocyclic substituent of the substrate, induced remarkable changes in the substrate specificities. In particular, the substrate specificity of mutant F159A toward aromatic substrate hydroxyphenylhydantoin (HPH) was enhanced by approximately 200-fold compared with that of the wild-type enzyme. Saturation mutagenesis at position 159 revealed that k(cat) for aromatic substrates increased gradually as the size of the amino acid side chain decreased, and this seems to be due to reduced steric hindrance between the bulky exocyclic group of the substrate and the amino acid side chains. When site-directed random mutagenesis of residues 63 and 65 was conducted with the wild type and mutant F159A, the selected enzymes (M63F/L65V and L65F/F159A) exhibited approximately 10-fold higher k(cat) values for HPH than the wild-type counterpart, which is likely to result from reorganization of the active site for efficient turnover. These results indicate that the amino acid residues of SGLs forming the substrate binding pocket are critical for the substrate specificity of D-hydantoinase, and the results also imply that substrate specificities of cyclic amidohydrolase family enzymes can be modulated by rational design of these SGLs.     

Mutant cholinesterases possessing enhanced capacity for reactivation of their phosphonylated conjugates

Selective mutants of mouse acetylcholinesterase (AChE; EC 3.1.1.7) phosphonylated with chiral S(P)- and R(P)-cycloheptyl, -3,3-dimethylbutyl, and -isopropyl methylphosphonyl thiocholines were subjected to reactivation by the oximes HI-6 and 2-PAM and their reactivation kinetics compared with wild-type AChE and butyrylcholinesterase (EC 3.1.1.8). Mutations in the choline binding site (Y337A, Y337A/F338A) or combined with acyl pocket mutations (F295L/Y337A, F297I/Y337A, F295L/F297I/Y337A) were employed to enlarge active center gorge dimensions. HI-6 was more efficient than 2-PAM (up to 29000 times) as a reactivator of S(P)-phosphonates (k(r) ranged from 50 to 13000 min(-1) M(-1)), while R(P) conjugates were reactivated by both oximes at similar, but far slower, rates (k(r) < 10 min(-1) M(-1)). The Y337A substitution accelerated all reactivation rates over the wild-type AChE and enabled reactivation even of R(P)-cycloheptyl and R(P)-3,3-dimethylbutyl conjugates that when formed in wild-type AChE are resistant to reactivation. When combined with the F295L or F297I mutations in the acyl pocket, the Y337A mutation showed substantial enhancements of reactivation rates of the S(P) conjugates. The greatest enhancement of 120-fold was achieved with HI-6 for the F295L/Y337A phosphonylated with the most bulky alkoxy moiety, S(P)-cycloheptyl methylphosphonate. This significant enhancement is likely a direct consequence of simultaneously increasing the dimensions of both the choline binding site and the acyl pocket. The increase in dimensions allows for optimizing the angle of oxime attack in the spatially impacted gorge as suggested from molecular modeling. Rates of reactivation reach values sufficient for consideration of mixtures of a mutant enzyme and an oxime as a scavenging strategy in protection and treatment of organophosphate exposure.     

Critical amino acids in phosphodiesterase-5 catalytic site that provide for high-affinity interaction with cyclic guanosine monophosphate and inhibitors

The molecular bases for phosphodiesterase 5 (PDE5) catalytic-site affinity for cyclic guanosine monophosphate (cGMP) and potency of inhibitors are poorly understood. Cocrystal structures of PDE5 catalytic (C) domain with inhibitors reveal a hydrogen bond and hydrophobic interactions with Tyr-612, hydrogen bonds with Gln-817, a hydrophobic clamp formed by Phe-820 and Val-782, and contacts with His-613, Leu-765, and Phe-786 [Sung et al. (2003) Nature 425, 98-102; Huai et al. (2004) J. Biol. Chem. 279, 13095-13101]. Present results of point mutations of full-length PDE5 showed that maximum catalysis was decreased 2650-fold in H613A and 55-fold in F820A. Catalytic-site affinities for cGMP, vardenafil, sildenafil, tadalafil, or 3-isobutyl-1-methylxanthine (IBMX) were respectively weakened 14-, 123-, 30-, 51-, and 43-fold for Y612A; 63-, 511-, 43-, 95- and 61-fold for Q817A; and 59-, 448-, 71-, 137-, and 93-fold for F820A. The data indicate that these three amino acids are major determinants of affinity for cGMP and potency of selective and nonselective inhibitors, and that higher vardenafil potency over sildenafil and tadalafil results from stronger contacts with Tyr-612, Gln-817, and Phe-820. Affinity of V782A for cGMP, vardenafil, sildenafil, tadalafil, or IBMX was reduced 5.5-, 23-, 10-, 3-, and 12-fold, respectively. Change in affinity for cGMP, vardenafil, sildenafil, or IBMX in Y612F, H613A, L765A, or F786A was less, but affinity of H613A or F786A for tadalafil was weakened 37- and 17-fold, respectively. The results quantify the role of PDE5 catalytic-site residues for cGMP and inhibitors, indicate that Tyr-612, Gln-817, and Phe-820 are the most important cGMP or inhibitor contacts studied, and identify residues that contribute to selectivity among different classes of inhibitors.     

Role of hydrogen bonds in the reaction mechanism of chalcone isomerase

In flavonoid, isoflavonoid, and anthocyanin biosynthesis, chalcone isomerase (CHI) catalyzes the intramolecular cyclization of chalcones into (S)-flavanones with a second-order rate constant that approaches the diffusion-controlled limit. The three-dimensional structures of alfalfa CHI complexed with different flavanones indicate that two sets of hydrogen bonds may possess critical roles in catalysis. The first set of interactions includes two conserved amino acids (Thr48 and Tyr106) that mediate a hydrogen bond network with two active site water molecules. The second set of hydrogen bonds occurs between the flavanone 7-hydroxyl group and two active site residues (Asn113 and Thr190). Comparison of the steady-state kinetic parameters of wild-type and mutant CHIs demonstrates that efficient cyclization of various chalcones into their respective flavanones requires both sets of contacts. For example, the T48A, T48S, Y106F, N113A, and T190A mutants exhibit 1550-, 3-, 30-, 7-, and 6-fold reductions in k(cat) and 2-3-fold changes in K(m) with 4,2',4'-trihydroxychalcone as a substrate. Kinetic comparisons of the pH-dependence of the reactions catalyzed by wild-type and mutant enzymes indicate that the active site hydrogen bonds contributed by these four residues do not significantly alter the pK(a) of the intramolecular cyclization reaction. Determinations of solvent kinetic isotope and solvent viscosity effects for wild-type and mutant enzymes reveal a change from a diffusion-controlled reaction to one limited by chemistry in the T48A and Y106F mutants. The X-ray crystal structures of the T48A and Y106F mutants support the assertion that the observed kinetic effects result from the loss of key hydrogen bonds at the CHI active site. Our results are consistent with a reaction mechanism for CHI in which Thr48 polarizes the ketone of the substrate and Tyr106 stabilizes a key catalytic water molecule. Hydrogen bonds contributed by Asn113 and Thr190 provide additional stabilization in the transition state. Conservation of these residues in CHIs from other plant species implies a common reaction mechanism for enzyme-catalyzed flavanone formation in all plants.     

Identification of overlapping but distinct cAMP and cGMP interaction sites with cyclic nucleotide phosphodiesterase 3A by site-directed mutagenesis and molecular modeling based on crystalline PDE4B

Cyclic nucleotide phosphodiesterase 3A (PDE3A) hydrolyzes cAMP to AMP, but is competitively inhibited by cGMP due to a low k(cat) despite a tight K(m). Cyclic AMP elevation is known to inhibit all pathways of platelet activation, and thus regulation of PDE3 activity is significant. Although cGMP elevation will inhibit platelet function, the major action of cGMP in platelets is to elevate cAMP by inhibiting PDE3A. To investigate the molecular details of how cGMP, a similar but not identical molecule to cAMP, behaves as an inhibitor of PDE3A, we constructed a molecular model of the catalytic domain of PDE3A based on homology to the recently determined X-ray crystal structure of PDE4B. Based on the excellent fit of this model structure, we mutated nine amino acids in the putative catalytic cleft of PDE3A to alanine using site-directed mutagenesis. Six of the nine mutants (Y751A, H840A, D950A, F972A, Q975A, and F1004A) significantly decreased catalytic efficiency, and had k(cat)/K(m) less than 10% of the wild-type PDE3A using cAMP as substrate. Mutants N845A, F972A, and F1004A showed a 3- to 12-fold increase of K(m) for cAMP. Four mutants (Y751A, H840A, D950A, and F1004A) had a 9- to 200-fold increase of K(i) for cGMP in comparison to the wild-type PDE3A. Studies of these mutants and our previous study identified two groups of amino acids: E866 and F1004 contribute commonly to both cAMP and cGMP interactions while N845, E971, and F972 residues are unique for cAMP and the residues Y751, H836, H840, and D950 interact with cGMP. Therefore, our results provide biochemical evidence that cGMP interacts with the active site residues differently from cAMP.     

Biochemical characterization and structural analysis of a highly proficient cocaine esterase

The bacterial cocaine esterase, cocE, hydrolyzes cocaine faster than any other reported cocaine esterase. Hydrolysis of the cocaine benzoyl ester follows Michaelis-Menten kinetics with k(cat) = 7.8 s(-1) and K(M) = 640 nM. A similar rate is observed for hydrolysis of cocaethylene, a more potent cocaine metabolite that has been observed in patients who concurrently abuse cocaine and alcohol. The high catalytic proficiency, lack of observable product inhibition, and ability to hydrolyze both cocaine and cocaethylene make cocE an attractive candidate for rapid cocaine detoxification in an emergency setting. Recently, we determined the crystal structure of this enzyme, and showed that it is a serine carboxylesterase, with a catalytic triad formed by S117, H287, and D259 within a hydrophobic active site, and an oxyanion hole formed by the backbone amide of Y118 and the Y44 hydroxyl. The only enzyme previously known to use a Tyr side chain to form the oxyanion hole is prolyl oligopeptidase, but the Y44F mutation of cocE has a more deleterious effect on the specificity rate constant (k(cat)/K(M)) than the analogous Y473F mutation of prolyl oligopeptidase. Kinetic studies on a series of cocE mutants both validate the proposed mechanism, and reveal the relative contributions of active site residues toward substrate recognition and catalysis. Inspired by the anionic binding pocket of the cocaine binding antibody GNC92H2, we found that a Q55E mutation within the active site of cocE results in a modest (2-fold) improvement in K(M), but a 14-fold loss of k(cat). The pH rate profile of cocE was fit to the ionization of two groups (pK(a1) = 7.7; pK(a2) = 10.4) that likely represent titration of H287 and Y44, respectively. We also describe the crystal structures of both S117A and Y44F mutants of cocE. Finally, urea denaturation studies of cocE by fluorescence and circular dichroism show two unfolding transitions (0.5-0.6 M and 3.2-3.7 M urea), with the first transition likely representing pertubation of the active site.     

Studies on Phe-228 and Leu-307 recombinant mutants of porcine kidney D-amino acid oxidase: expression, purification, and characterization.

Two recombinant mutants of porcine kidney D-amino acid oxidase [EC 1.4.3.3, DAO], in which Tyr(228) and His(307) are replaced with Phe and Leu, respectively, have been expressed in Escherichia coli and purified to apparent homogeneity. The molecular size and amino-terminal sequence of the two mutants were the same as those of the native DAO. Kinetic analysis revealed that the Michaelis constants of the Phe-228 and Leu-307 mutants for D-alanine were 71- and 10-fold and the inhibition constants for benzoate, a potent competitive inhibitor, were 1,189- and 18-fold greater than those of the native DAO, respectively. The maximum velocities of the Phe-228 and Leu-307 mutants were 66 and 58% that of the native DAO. The kinetically estimated dissociation constant of the Leu-307 mutant for FAD was 28-fold greater than that of the native DAO, whereas the value of the Phe-228 mutant was comparable to that of the native DAO. The Leu-307 mutant and the recombinant wild-type DAO were inactivated by D-propargylglycine (D-PG), a suicide substrate. However, the Phe-228 mutant was resistant to the inactivation. Absorption peaks of the Phe-228 mutant were blue-shifted about 10 nm from the corresponding peaks of the wild-type DAO, and the oxidized form was fully reduced by D-alanine without appearance of the purple intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for electron equilibrium between the two hemes bL in the dimeric cytochrome bc1 complex

Structural analysis of the dimeric mitochondrial cytochrome bc1 complex suggests that electron transfer between inter-monomer hemes bL-bL may occur during bc1 catalysis. Such electron transfer may be facilitated by the aromatic pairs present between the two bL hemes in the two symmetry-related monomers. To test this hypothesis, R. sphaeroides mutants expressing His6-tagged bc1 complexes with mutations at three aromatic residues (Phe-195, Tyr-199, and Phe-203), located between two bL hemes, were generated and characterized. All three mutants grew photosynthetically at a rate comparable to that of wild-type cells. The bc1 complexes prepared from mutants F195A, Y199A, and F203A have, respectively, 78%, 100%, and 100% of ubiquinol-cytochrome c reductase activity found in the wild-type complex. Replacing the Phe-195 of cytochrome b with Tyr, His, or Trp results in mutant complexes (F195Y, F195H, or F195W) having the same ubiquinol-cytochrome c reductase activity as the wild-type. These results indicate that the aromatic group at position195 of cytochrome b is involved in electron transfer reactions of the bc1 complex. The rate of superoxide anion (O2*) generation, measured by the chemiluminescence of 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha]pyrazin-3-one hydrochloride-O2* adduct during oxidation of ubiquinol, is 3 times higher in the F195A complex than in the wild-type or mutant complexes Y199A or F203A. This supports the idea that the interruption of electron transfer between the two bL hemes enhances electron leakage to oxygen and thus decreases the ubiquinol-cytochrome c reductase activity.