Arg-158 is critical in both binding the substrate and stabilizing the transition-state oxyanion for the enzymatic reaction of malonamidase E2

Malonamidase E2 (MAE2) from Bradyrhizobium japonicum is an enzyme that hydrolyzes malonamate to malonate and has a Ser-cis-Ser-Lys catalytic triad at the active site. The crystal structures of wild type and mutant MAE2 exhibited that the guanido group of Arg-158 could be involved in the binding of malonamate in which the negative charge of the carboxyl group could destabilize a negatively charged transition-state oxyanion in the enzymatic reaction. In an attempt to elucidate the specific roles of Arg-158, site-directed mutants, R158Q, R158E, and R158K, were prepared (see Table 1). The crystal structure of R158Q determined at 2.2 Angstrom resolution showed that the guanido group of Arg-158 was important for the substrate binding with the marginal structural change upon the mutation. The k(cat) value of R158Q significantly decreased by over 1500-fold and the catalytic activity of R158E could not be detected. The k(cat) value of R158K was similar to that of the wild type with the K(m) value drastically increased by 100-fold, suggesting that Lys-158 of R158K can stabilize the negative charge of the carboxylate in the substrate to some extent and contribute to the stabilization of the transition-state oxyanion, but a single amine group of Lys-158 in R158K could not precisely anchor the carboxyl group of malonamate compared with the guanido group of Arg-158. Our kinetic and structural evidences demonstrate that Arg-158 in MAE2 should be critical to both binding the substrate and stabilizing the transition-state oxyanion for the catalytic reaction of MAE2.     

Role of arginine 59 in the gamma-class carbonic anhydrases.

The functional role of the highly conserved active site Arg 59 in the prototype of the gamma-class carbonic anhydrase Cam (carbonic anhydrase from Methanosarcina thermophila) was investigated. Variants (R59A, -C, -E, -H, -K, -M, and -Q) were prepared by site-directed mutagenesis and characterized by size exclusion chromatography (SEC), circular dichroism (CD) spectroscopy, and stopped-flow kinetic analyses. CD spectra indicated similar secondary structures for the wild type and the R59A and -K variants, independent of nondenaturing concentrations of guanidine hydrochloride (GdnHCl). SEC indicated that all variants purified as homotrimers like the wild type. SEC also revealed that the R59A and -K variants unfolded at > or = 1.5 M GdnHCl, compared to 3.0 M GdnHCl for the wild type. These results indicate that Arg 59 contributes to the thermodynamic stability of the Cam trimer. The R59K variant had k(cat) and k(cat)/K(m) values that were 8 and 5% of the wild-type values, respectively, while all other variants had k(cat) and k(cat)/K(m) values 10-100-fold lower than those of the wild type. The R59A, -C, -E, -M, and -Q variants exhibited 4-63-fold increases in k(cat) and 9-120-fold increases in k(cat)/K(m) upon addition of 100 mM GdnHCl, with the largest increases observed for the R59A variant, which was comparable to the R59K variant. The kinetic results indicate that a positive charge at position 59 is essential for the CO(2) hydration step of the overall catalytic mechanism.     

Discrimination of cognate and noncognate substrates at the active site of class I lysyl-tRNA synthetase

The aminoacyl-tRNA synthetases are divided into two unrelated structural classes, with lysyl-tRNA synthetase (LysRS) being the only enzyme represented in both classes. On the basis of the structure of l-lysine complexed with Pyrococcus horikoshii class I LysRS (LysRS1) and homology to glutamyl-tRNA synthetase (GluRS), residues implicated in amino acid recognition and noncognate substrate discrimination were systematically replaced in Borrelia burgdorferi LysRS1. The catalytic efficiency of steady-state aminoacylation (k(cat)/K(M)) with lysine by LysRS1 variants fell by 1-4 orders of magnitude compared to that of the wild type. Disruption of putative hydrogen bonding interactions through replacement of G29, T31, and Y269 caused up to 1500-fold reductions in k(cat)/K(M), similar to changes previously observed for comparable variants of class II LysRS (LysRS2). Replacements of W220 and H242, both of which are implicated in hydrophobic interactions with the side chain of lysine, resulted in more dramatic changes with up to 40000-fold reductions in k(cat)/K(M) observed. This indicates that the more compact LysRS1 active site employs both electrostatic and hydrophobic interactions during lysine discrimination, explaining the ability of LysRS1 to discriminate against noncognate substrates accepted by LysRS2. Several of the LysRS1 variants were found to be more specific than the wild type with respect to noncognate amino acid recognition but less efficient in cognate aminoacylation. This indicates that LysRS1 compromises between efficient catalysis and substrate discrimination, in contrast to LysRS2 which is considerably more effective in catalysis but is less specific than its class I counterpart.     

Electrostatic environment surrounding the activation loop phosphotyrosine in the oncoprotein v-Fps

Autophosphorylation of Tyr-1073 in the activation loop of the oncoprotein v-Fps enhances the phosphoryl transfer reaction without influencing substrate, ATP, or metal ion binding affinities [Saylor, P., et al. (1998) Biochemistry 37, 17875-17881]. A structural model of v-Fps, generated from the insulin receptor, indicates that pTyr-1073 chelates two arginines. Mutation of these residues to alanine (R1042A and R1066A) results in weakly phosphorylated enzymes, indicating that one electropositive center is insufficient for attaining maximum loop phosphorylation and concomitant high catalytic activity. While the turnover rate for R1066A is similar to that for a mutant lacking a phosphorylatable residue in the activation loop, the rate for R1042A is 50-fold slower. While solvent perturbation studies suggest that the former is due to a slow phosphoryl transfer step, the latter effect results from a slow conformational change in the mutant, potentially linked to motions in the catalytic loop. Binding of a stoichiometric quantity of Mg(2+) is essential for ATP binding and catalysis, while binding of an additional Mg(2+) ion activates further the wild-type enzyme. The affinity of the R1066A enzyme for the second Mg(2+) ion is 23-fold higher than that of the phosphorylated or unphosphorylated form of wild-type v-Fps, with substrate binding unaffected. Conversely, the affinity of R1066A for a substrate mimic lacking a phosphorylation site is 12-fold higher than that for the phosphorylated or unphosphorylated form of wild-type v-Fps, with binding of the second Mg(2+) ion unaffected. A comparison of these enzyme-independent parameters indicates that Arg-1042 and Arg-1066 induce strain in the active site in the repressed form of the enzyme. While this strain is not relieved in the phosphorylated form, the improvements in catalysis in activated v-Fps compensate for reduced metal and substrate binding affinities.     

Enhancement, relaxation, and reversal of the stereoselectivity for phosphotriesterase by rational evolution of active site residues

The factors that govern the substrate reactivity and stereoselectivity of phosphotriesterase (PTE) toward organophosphotriesters containing various combinations of methyl, ethyl, isopropyl, and phenyl substituents at the phosphorus center were determined by systematic alterations in the dimensions of the active site. The wild type PTE prefers the S(P)-enantiomers over the corresponding R(P)-enantiomers by factors ranging from 10 to 90. Enlargement of the small subsite of PTE with the substitution of glycine and alanine residues for Ile-106, Phe-132, and/or Ser-308 resulted in significant improvements in k(cat)/K(a) for the R(P)-enantiomers of up to 2700-fold but had little effect on k(cat)/K(a) for the corresponding S(P)-enantiomers. The kinetic preferences for the S(P)-enantiomers were thus relaxed without sacrificing the inherent catalytic activity of the wild type enzyme. A reduction in the size of the large subsite with the mutant H257Y resulted in a reduction in k(cat)/K(a) for the S(P)-enantiomers, while the values of k(cat)/K(a) for the R(P)-enantiomers were essentially unchanged. The initial stereoselectivity observed with the wild type enzyme toward the chiral substrate library was significantly reduced with the H257Y mutant. Simultaneous alternations in the sizes of the large and small subsites resulted in the complete reversal of the chiral specificity. With this series of mutants, the R(P)-enantiomers were preferred as substrates over the corresponding S(P)-enantiomers by up to 500-fold. These results have demonstrated that the stereochemical determinants for substrate hydrolysis by PTE can be systematically altered through a rational reconstruction of the dimensions of the active site.     

Chemical rescue of a mutant protein-tyrosine kinase

Protein-tyrosine kinases contain a catalytic loop Arg residue located either two or four positions downstream of a highly conserved Asp residue. In this study, the role of this Arg (Arg-318) in the protein-tyrosine kinase C-terminal Src kinase (Csk) was investigated. The observed k(cat) for phosphorylation of the random copolymer poly(Glu,Tyr) substrate by Csk R318A is approximately 3000-fold smaller compared with that of wild type Csk, whereas the K(m) values for ATP and poly(Glu,Tyr) are only mildly affected. The k(cat) value for poly(Glu,Tyr) phosphorylation by the Csk double mutant A316R,R318A is 100-fold greater than the k(cat) value for the single R318A mutant, suggesting that an Arg positioned at the alternative location fulfills a similar function as in wild type. Csk R318A kinase activity can also be partially recovered by several exogenous small molecules including guanidinium and imidazole. These molecules contain key features whose roles in catalysis can be rationalized from a known x-ray structure of the insulin receptor tyrosine kinase. Imidazole is the best of these activators, enhancing phosphorylation rates by Csk R318A up to 100-fold for poly(Glu,Tyr) and significantly stimulating Csk R318A phosphorylation of the physiologic substrate Src. This chemical rescue of mutant protein kinase activity might find applications in cell signal transduction experiments.     

Effects of mutations of the active site arginine residues in 4-oxalocrotonate tautomerase on the pKa values of active site residues and on the pH dependence of catalysis.

The unusually low pK(a) value of the general base catalyst Pro-1 (pK(a) = 6.4) in 4-oxalocrotonate tautomerase (4-OT) has been ascribed to both a low dielectric constant at the active site and the proximity of the cationic residues Arg-11 and Arg-39 [Stivers, J. T., Abeygunawardana, C., Mildvan, A. S., Hajipour, G., and Whitman, C. P. (1996) Biochemistry 35, 814-823]. In addition, the pH-rate profiles in that study showed an unidentified protonated group essential for catalysis with a pK(a) of 9.0. To address these issues, the pK(a) values of the active site Pro-1 and lower limit pK(a) values of arginine residues were determined by direct (15)N NMR pH titrations. The pK(a) values of Pro-1 and of the essential acid group were determined independently from pH-rate profiles of the kinetic parameters of 4-OT in arginine mutants of 4-OT and compared with those of wild type. The chemical shifts of all of the Arg Nepsilon resonances in wild-type 4-OT and in the R11A and R39Q mutants were found to be independent of pH over the range 4.9-9.7, indicating that no arginine is responsible for the kinetically determined pK(a) of 9.0 for an acidic group in free 4-OT. With the R11A mutant, where k(cat)/K(m) was reduced by a factor of 10(2.9), the pK(a) of Pro-1 was not significantly altered from that of the wild-type enzyme (pK(a) = 6.4 +/- 0.2) as revealed by both direct (15)N NMR titration (pK(a) = 6.3 +/- 0.1) and the pH dependence of k(cat)/K(m) (pK(a) = 6.4 +/- 0.2). The pH-rate profiles of both k(cat)/K(m) and k(cat) for the reaction of the R11A mutant with the dicarboxylate substrate, 2-hydroxymuconate, showed humps, i.e., sharply defined maxima followed by nonzero plateaus. The humps disappeared in the reaction with the monocarboxylate substrate, 2-hydroxy-2,4-pentadienoate, indicating that, unlike the wild-type enzyme which reacts only with the dianionic form of the dicarboxylic substrate, the R11A mutant reacts with both the 6-COOH and 6-COO(-) forms, with the 6-COOH form being 12-fold more active. This reversal in the preferred ionization state of the 6-carboxyl group of the substrate that occurs upon mutation of Arg-11 to Ala provides strong evidence that Arg-11 interacts with the 6-carboxylate of the substrate. In the R39Q mutant, where k(cat)/K(m) was reduced by a factor of 10(3), the kinetically determined pK(a) value for Pro-1 was 4.6 +/- 0.2, while the ionization of Pro-1 showed negative cooperativity with an apparent pK(a) of 7.1 +/- 0.1 determined by 1D (15)N NMR. From the Hill coefficient of 0.54, it can be shown that the apparent pK(a) value of 7.1 could result most simply from the averaging of two limiting pK(a) values of 4.6 and 8.2. Mutation of Arg-39, by altering the structure of the beta-hairpin which covers the active site, could result in an increase in the solvent exposure of Pro-1, raising its upper limit pK(a) value to 8.2. In the R39A mutant, the kinetically determined pK(a) of Pro-1 was also low, 5.0 +/- 0.2, indicating that in both the R39Q and R39A mutants, only the sites with low pK(a) values were kinetically operative. With the fully active R61A mutant, the kinetically determined pK(a) of Pro-1 (pK(a) = 6.5 +/- 0.2) agreed with that of wild-type 4-OT. It is concluded that the unusually low pK(a) of Pro-1 shows little contribution from electrostatic effects of the nearby cationic Arg-11, Arg-39, and Arg-61 residues but results primarily from a site of low local dielectric constant.     

A structure-function study of a proton transport pathway in the gamma-class carbonic anhydrase from Methanosarcina thermophila

Four glutamate residues in the prototypic gamma-class carbonic anhydrase from Methanosarcina thermophila (Cam) were characterized by site-directed mutagenesis and chemical rescue studies. Alanine substitution indicated that an external loop residue, Glu 84, and an internal active site residue, Glu 62, are both important for CO(2) hydration activity. Two other external loop residues, Glu 88 and Glu 89, are less important for enzyme function. The two E84D and -H variants exhibited significant activity relative to wild-type activity in pH 7.5 MOPS buffer, suggesting that the original glutamate residue could be substituted with other ionizable residues with similar pK(a) values. The E84A, -C, -K, -Q, -S, and -Y variants exhibited large decreases in k(cat) values in pH 7.5 MOPS buffer, but only exhibited small changes in k(cat)/K(m). These same six variants were all chemically rescued by pH 7.5 imidazole buffer, with 23-46-fold increases in the apparent k(cat). These results are consistent with Glu 84 functioning as a proton shuttle residue. The E62D variant exhibited a 3-fold decrease in k(cat) and a 2-fold decrease in k(cat)/K(m) relative to those of the wild type in pH 7.5 MOPS buffer, while other substitutions (E62A, -C, -H, -Q, -T, and -Y) resulted in much larger decreases in both k(cat) and k(cat)/K(m). Imidazole did not significantly increase the k(cat) values and slightly decreased the k(cat)/K(m) values of most of the Glu 62 variants. These results indicate a primary preference for a carboxylate group at position 62, and support a proposed catalytic role for residue Glu 62 in the CO(2) hydration step, but do not definitively establish its role in the proton transport step.     

Roles of the conserved aspartate and arginine in the catalytic mechanism of an archaeal beta-class carbonic anhydrase

The roles of an aspartate and an arginine, which are completely conserved in the active sites of beta-class carbonic anhydrases, were investigated by steady-state kinetic analyses of replacement variants of the beta-class enzyme (Cab) from the archaeon Methanobacterium thermoautotrophicum. Previous kinetic analyses of wild-type Cab indicated a two-step zinc-hydroxide mechanism of catalysis in which the k(cat)/K(m) value depends only on the rate constants for the CO(2) hydration step, whereas k(cat) also depends on rate constants from the proton transfer step (K. S. Smith, N. J. Cosper, C. Stalhandske, R. A. Scott, and J. G. Ferry, J. Bacteriol. 182:6605-6613, 2000). The recently solved crystal structure of Cab shows the presence of a buffer molecule within hydrogen bonding distance of Asp-34, implying a role for this residue in the proton transport step (P. Strop, K. S. Smith, T. M. Iverson, J. G. Ferry, and D. C. Rees, J. Biol. Chem. 276:10299-10305, 2001). The k(cat)/K(m) values of Asp-34 variants were decreased relative to those of the wild type, although not to an extent which supports an essential role for this residue in the CO(2) hydration step. Parallel decreases in k(cat) and k(cat)/K(m) values for the variants precluded any conclusions regarding a role for Asp-34 in the proton transfer step; however, the k(cat) of the D34A variant was chemically rescued by replacement of 2-(N-morpholino)propanesulfonic acid buffer with imidazole at pH 7.2, supporting a role for the conserved aspartate in the proton transfer step. The crystal structure of Cab also shows Arg-36 with two hydrogen bonds to Asp-34. Arg-36 variants had both k(cat) and k(cat)/K(m) values that were decreased at least 250-fold relative to those of the wild type, establishing an essential function for this residue. Imidazole was unable to rescue the k(cat) of the R36A variant; however, partial rescue of the kinetic parameter was obtained with guanidine-HCl indicating that the guanido group of this residue is important.     

Kinetic, stereochemical, and structural effects of mutations of the active site arginine residues in 4-oxalocrotonate tautomerase.

Three arginine residues (Arg-11, Arg-39, Arg-61) are found at the active site of 4-oxalocrotonate tautomerase in the X-ray structure of the affinity-labeled enzyme [Taylor, A. B., Czerwinski, R. M., Johnson, R. M., Jr., Whitman, C. P., and Hackert, M. L. (1998) Biochemistry 37, 14692-14700]. The catalytic roles of these arginines were examined by mutagenesis, kinetic, and heteronuclear NMR studies. With a 1,6-dicarboxylate substrate (2-hydroxymuconate), the R61A mutation showed no kinetic effects, while the R11A mutation decreased k(cat) 88-fold and increased K(m) 8.6-fold, suggesting both binding and catalytic roles for Arg-11. With a 1-monocarboxylate substrate (2-hydroxy-2,4-pentadienoate), no kinetic effects of the R11A mutation were found, indicating that Arg-11 interacts with the 6-carboxylate of the substrate. The stereoselectivity of the R11A-catalyzed protonation at C-5 of the dicarboxylate substrate decreased, while the stereoselectivity of protonation at C-3 of the monocarboxylate substrate increased in comparison with wild-type 4-OT, indicating the importance of Arg-11 in properly orienting the dicarboxylate substrate by interacting with the charged 6-carboxylate group. With 2-hydroxymuconate, the R39A and R39Q mutations decreased k(cat) by 125- and 389-fold and increased K(m) by 1.5- and 2.6-fold, respectively, suggesting a largely catalytic role for Arg-39. The activity of the R11A/R39A double mutant was at least 10(4)-fold lower than that of the wild-type enzyme, indicating approximate additivity of the effects of the two arginine mutants on k(cat). For both R11A and R39Q, 2D (1)H-(15)N HSQC and 3D (1)H-(15)N NOESY-HSQC spectra showed chemical shift changes mainly near the mutated residues, indicating otherwise intact protein structures. The changes in the R39Q mutant were mainly in the beta-hairpin from residues 50 to 57 which covers the active site. HSQC titration of R11A with the substrate analogue cis, cis-muconate yielded a K(d) of 22 mM, 37-fold greater than the K(d) found with wild-type 4-OT (0.6 mM). With the R39Q mutant, cis, cis-muconate showed negative cooperativity in active site binding with two K(d) values, 3.5 and 29 mM. This observation together with the low K(m) of 2-hydroxymuconate (0.47 mM) suggests that only the tight binding sites function catalytically in the R39Q mutant. The (15)Nepsilon resonances of all six Arg residues of 4-OT were assigned, and the assignments of Arg-11, -39, and -61 were confirmed by mutagenesis. The binding of cis,cis-muconate to wild-type 4-OT upshifts Arg-11 Nepsilon (by 0.05 ppm) and downshifts Arg-39 Nepsilon (by 1.19 ppm), indicating differing electronic delocalizations in the guanidinium groups. A mechanism is proposed in which Arg-11 interacts with the 6-carboxylate of the substrate to facilitate both substrate binding and catalysis and Arg-39 interacts with the 1-carboxylate and the 2-keto group of the substrate to promote carbonyl polarization and catalysis, while Pro-1 transfers protons from C-3 to C-5. This mechanism, together with the effects of mutations of catalytic residues on k(cat), provides a quantitative explanation of the 10(7)-fold catalytic power of 4-OT. Despite its presence in the active site in the crystal structure of the affinity-labeled enzyme, Arg-61 does not play a significant role in either substrate binding or catalysis.     

The function of Arg-94 in the oxidation and decarboxylation of glutaryl-CoA by human glutaryl-CoA dehydrogenase

Glutaryl-CoA dehydrogenase catalyzes the oxidation and decarboxylation of glutaryl-CoA to crotonyl-CoA and CO(2). Inherited defects in the protein cause glutaric acidemia type I, a fatal neurologic disease. Glutaryl-CoA dehydrogenase is the only member of the acyl-CoA dehydrogenase family with a cationic residue, Arg-94, situated in the binding site of the acyl moiety of the substrate. Crystallographic investigations suggest that Arg-94 is within hydrogen bonding distance of the gamma-carboxylate of glutaryl-CoA. Substitution of Arg-94 by glycine, a disease-causing mutation, and by glutamine, which is sterically more closely related to arginine, reduced k(cat) of the mutant dehydrogenases to 2-3% of k(cat) of the wild type enzyme. K(m) of these mutant dehydrogenases for glutaryl-CoA increases 10- to 16-fold. The steady-state kinetic constants of alternative substrates, hexanoyl-CoA and glutaramyl-CoA, which are not decarboxylated, are modestly affected by the mutations. The latter changes are probably due to steric and polar effects. The dissociation constants of the non-oxidizable substrate analogs, 3-thiaglutaryl-CoA and acetoacetyl-CoA, are not altered by the mutations. However, abstraction of a alpha-proton from 3-thiaglutaryl-CoA, to yield a charge transfer complex with the oxidized flavin, is severely limited. In contrast, abstraction of the alpha-proton of acetoacetyl-CoA by Arg-94 --> Gln mutant dehydrogenase is unaffected, and the resulting enolate forms a charge transfer complex with the oxidized flavin. These experiments indicate that Arg-94 does not make a major contribution to glutaryl-CoA binding. However, the electric field of Arg-94 may stabilize the dianions resulting from abstraction of the alpha-proton of glutaryl-CoA and 3-thiaglutaryl-CoA, both of which contain gamma-carboxylates. It is also possible that Arg-94 may orient glutaryl-CoA and 3-thiaglutaryl-CoA for abstraction of an alpha-proton.     

Identification, characterization, and site-directed mutagenesis of recombinant pentachlorophenol 4-monooxygenase

In a previous study, we constructed a three-dimensional (3D) structure of pentachlorophenol 4-monooxygenase (PcpB). In this study, further analyses are performed to examine the important amino acid residues in the catalytic reaction by identification of the proteins with mass spectrometry, circular dichroism (CD) and UV spectrometry, and determination of kinetic parameters. Recombinant histidine-tagged PcpB protein was produced and shown to have a similar activity to the native protein. Mutant proteins of PcpB were then produced (F85A, Y216A, Y216F, R235A, R235E, R235K, Y397A and Y397F) on the basis of the proposed 3D structure. The CD spectra of the proteins showed that there were no major changes in the structures of the mutant proteins, with the exception of R235E. Steady-state kinetics showed a 20-fold reduction in k(cat)/K(m) and a ninefold increase in K(m) for Y216F and a threefold reduction in k(cat)/K(m) and a sixfold increase in K(m) for Y397F compared to the wild type. On the other hand, the value of k(cat)/K(m) of R235K mutant was the same as that of wild type. As a result, it was confirmed that Y216 and Y397 play an important role with respect to the recognition of the substrate.     

Catalytic role for arginine 188 in the C-C hydrolase catalytic mechanism for Escherichia coli MhpC and Burkholderia xenovorans LB400 BphD

The alpha/beta-hydrolase superfamily, comprised mainly of esterase and lipase enzymes, contains a family of bacterial C-C hydrolases, including MhpC and BphD which catalyze the hydrolytic C-C cleavage of meta-ring fission intermediates on the Escherichia coli phenylpropionic acid pathway and Burkholderia xenovorans LB400 biphenyl degradation pathway, respectively. Five active site amino acid residues (Arg-188, Asn-109, Phe-173, Cys-261, and Trp-264) were identified from sequence alignments that are conserved in C-C hydrolases, but not in enzymes of different function. Replacement of Arg-188 in MhpC with Gln and Lys led to 200- and 40-fold decreases, respectively, in k(cat); the same replacements for Arg-190 of BphD led to 400- and 700-fold decreases, respectively, in k(cat). Pre-steady-state kinetic analysis of the R188Q MhpC mutant revealed that the first step of the reaction, keto-enol tautomerization, had become rate-limiting, indicating that Arg-188 has a catalytic role in ketonization of the dienol substrate, which we propose is via substrate destabilization. Mutation of nearby residues Phe-173 and Trp-264 to Gly gave 4-10-fold reductions in k(cat) but 10-20-fold increases in K(m), indicating that these residues are primarily involved in substrate binding. The X-ray structure of a succinate-H263A MhpC complex shows concerted movements in the positions of both Phe-173 and Trp-264 that line the approach to Arg-188. Mutation of Asn-109 to Ala and His yielded 200- and 350-fold reductions, respectively, in k(cat) and pre-steady-state kinetic behavior similar to that of a previous S110A mutant, indicating a role for Asn-109 is positioning the active site loop containing Ser-110. The catalytic role of Arg-188 is rationalized by a hydrogen bond network close to the C-1 carboxylate of the substrate, which positions the substrate and promotes substrate ketonization, probably via destabilization of the bound substrate.     

Evolution of enzymatic activity in the tautomerase superfamily: mechanistic and structural consequences of the L8R mutation in 4-oxalocrotonate tautomerase

4-Oxalocrotonate tautomerase (4-OT) and trans-3-chloroacrylic acid dehalogenase (CaaD) are members of the tautomerase superfamily, a group of structurally homologous proteins that share a beta-alpha-beta fold and a catalytic amino-terminal proline. 4-OT, from Pseudomonas putida mt-2, catalyzes the conversion of 2-oxo-4-hexenedioate to 2-oxo-3-hexenedioate through the dienol intermediate 2-hydroxymuconate in a catabolic pathway for aromatic hydrocarbons. CaaD, from Pseudomonas pavonaceae 170, catalyzes the hydrolytic dehalogenation of trans-3-chloroacrylate in the trans-1,3-dichloropropene degradation pathway. Both reactions may involve an arginine-stabilized enediolate intermediate, a capability that may partially account for the low-level CaaD activity of 4-OT. Two active-site residues in 4-OT, Leu-8 and Ile-52, have now been mutated to the positionally conserved and catalytic ones in CaaD, alphaArg-8, and alphaGlu-52. The L8R and L8R/I52E mutants show improved CaaD activity (50- and 32-fold increases in k(cat)/K(m), respectively) and diminished 4-OT activity (5- and 1700-fold decreases in k(cat)/K(m), respectively). The increased efficiency of L8R-4-OT for the CaaD reaction stems primarily from an 8.8-fold increase in k(cat), whereas that of the L8R/I52E mutant is due largely to a 23-fold decrease in K(m). The presence of the additional arginine residue in the active site of L8R-4-OT does not alter the pK(a) of the Pro-1 amino group from that measured for the wild type (6.5 +/- 0.1 versus 6.4 +/- 0.2). Moreover, the crystal structure of L8R-4-OT is comparable to that of the wild type. Hence, the enhanced CaaD activity of L8R-4-OT is likely due to the additional arginine residue that can participate in substrate binding and/or stabilization of the putative enediolate intermediate. The results also suggest that the evolution of new functions within the tautomerase superfamily could be quite facile, requiring only a few strategically placed active-site mutations.     

Catalytic reaction pathway for the mitogen-activated protein kinase ERK2

The structural, functional, and regulatory properties of the mitogen-activated protein kinases (MAP kinases) have long attracted considerable attention owing to the critical role that these enzymes play in signal transduction. While several MAP kinase X-ray crystal structures currently exist, there is by comparison little mechanistic information available to correlate the structural data with the known biochemical properties of these molecules. We have employed steady-state kinetic and solvent viscosometric techniques to characterize the catalytic reaction pathway of the MAP kinase ERK2 with respect to the phosphorylation of a protein substrate, myelin basic protein (MBP), and a synthetic peptide substrate, ERKtide. A minor viscosity effect on k(cat) with respect to the phosphorylation of MBP was observed (k(cat) = 10 +/- 2 s(-1), k(cat)(eta) = 0.18 +/- 0.05), indicating that substrate processing occurs via slow phosphoryl group transfer (12 +/- 4 s(-1)) followed by the faster release of products (56 +/- 4 s(-1)). At an MBP concentration extrapolated to infinity, no significant viscosity effect on k(cat)/K(m(ATP)) was observed (k(cat)/K(m(ATP)) = 0.2 +/- 0.1 microM(-1) s(-1), k(cat)/K(m(ATP))(eta) = -0.08 +/- 0.04), consistent with rapid-equilibrium binding of the nucleotide. In contrast, at saturating ATP, a full viscosity effect on k(cat)/K(m) for MBP was apparent (k(cat)/K(m(MBP)) = 2.4 +/- 1 microM(-1) s(-1), k(cat)/K(m(MBP))(eta) = 1.0 +/- 0.1), while no viscosity effect was observed on k(cat)/K(m) for the phosphorylation of ERKtide (k(cat)/K(m(ERKtide)) = (4 +/- 2) x 10(-3) microM(-1) s(-1), k(cat)/K(m(ERKtide))(eta) = -0.02 +/- 0.02). This is consistent with the diffusion-limited binding of MBP, in contrast to the rapid-equilibrium binding of ERKtide, to form the ternary Michaelis complex. Calculated values for binding constants show that the estimated value for K(d(MBP)) (/= 1.5 mM). The dramatically higher catalytic efficiency of MBP in comparison to that of ERKtide ( approximately 600-fold difference) is largely attributable to the slow dissociation rate of MBP (/=56 s(-1)), from the ERK2 active site.     

Coenzyme specificity of human monomeric carbonyl reductase: contribution of Lys-15, Ala-37 and Arg-38

Short-chain dehydrogenases/reductases catalyze the oxidoreduction of alcohol and carbonyl compounds using either NAD or NADPH as coenzyme. Structural analysis suggests that specificity for NADPH is conferred by two highly conserved basic residues in the N-terminal part of the peptide chain, whereas specificity for NAD correlates with the presence of an Asp adjacent to the position of the distal basic residue in NADP-dependent enzymes. We carried out site-directed mutagenesis of the two basic residues: Lys-15 and Arg-38, as well as of Ala-37 of human monomeric carbonyl reductase in order to investigate their contribution to coenzyme binding and specificity. Substitution of Lys-15 or Arg-38 by Gln and, even more pronounced Asp decreased the catalytic efficiency (k(cat)/K(m,NADPH)) by more than three orders of magnitude. Similarly, substitution of Asp for Ala-37 decreased k(cat)/K(m,NADPH) 1000-fold but had little effect on k(cat)/K(m,NADH). The results demonstrate the importance of basic residues at positions 15 and 38 and the absence of an acidic residue at position 37 for NADPH binding and catalysis.     

Identification of critical residues of choline kinase A2 from Caenorhabditis elegans

Choline kinase catalyzes the phosphorylation of choline by ATP, the first committed step in the CDP-choline pathway for phosphatidylcholine biosynthesis. To begin to elucidate the mechanism of catalysis by this enzyme, choline kinase A-2 from Caenorhabditis elegans was analyzed by systematic mutagenesis of highly conserved residues followed by analysis of kinetic and structural parameters. Specifically, mutants were analyzed with respect to K(m) and k(cat) values for each substrate and Mg(2+), inhibitory constants for Mg(2+) and Ca(2+), secondary structure as monitored by circular dichroism, and sensitivity to unfolding in guanidinium hydrochloride. The most severe impairment of catalysis occurred with the modification of Asp-255 and Asn-260, which are located in the conserved Brenner's phosphotransferase motif, and Asp-301 and Glu-303, in the signature choline kinase motif. For example, mutation of Asp-255 or Asp-301 to Ala eliminated detectable catalytic activity, and mutation of Asn-260 and Glu-303 to Ala decreased k(cat) by 300- and 10-fold, respectively. Additionally, the K(m) for Mg(2+) for mutants N260A and E303A was approximately 30-fold higher than that of wild type. Several other residues (Ser-86, Arg-111, Glu-125, and Trp-387) were identified as being important: Catalytic efficiencies (k(cat)/K(m)) for the enzymes in which these residues were mutated to Ala were reduced to 2-25% of wild type. The high degree of structural similarity among choline kinase A-2, aminoglycoside phosphotransferases, and protein kinases, together with the results from this mutational analysis, indicates it is likely that these conserved residues are located at the catalytic core of choline kinase.     

Autolytic processing at Glu586-Ser587 within the cysteine-rich domain of human adamalysin 19/disintegrin-metalloproteinase 19 is necessary for its proteolytic activity

We investigated the regulation of the proteolytic activity of human adamalysin 19 (a disintegrin and metalloproteinase 19, hADAM19). It was processed at Glu(586)(P1)-Ser(587)(P1') site in the cysteine-rich domain as shown by protein N-terminal sequencing. This truncation was autolytic as illustrated by its R199A/R200A or E346A mutation that prevented the zymogen activation by furin or abolished the catalytic activity. Reagents that block furin-mediated activation of pro-hADAM19, decRVKR-CMK, and brefeldin A abrogated this processing. The sizes of the side chains of the P1 and P1' residues are critical for the processing of hADAM19. The amount of processing product in the E586Q or S587A mutant with a side chain almost the same size as that in the wild type was almost equal. Conversely, very little processing was observed when the size of the side chain was changed significantly, such as in the E586A, E586G, or S587F mutants. Two mutants with presumably subtle structural distinctions from wild type hADAM19, E586D and S587T, displayed rare or little processing and had very low capacities to cleave alpha2-macroglobulin and a peptide substrate. Therefore, this processing is necessary for hADAM19 to exert its proteolytic activities. Moreover, a new peptide substrate, Ac-RPLE-SNAV, which is identical to the processing site sequence, was cleaved at the E-S bond by soluble hADAM19 containing the catalytic and disintegrin domains. This enzyme cleaved the substrate with K(m), k(cat), and k(cat)/K(m) of 2.0 mm, 2.4/min, and 1200 m(-1) min(-1), respectively, using a fluorescamine assay. Preliminary studies showed that a protein kinase C activator, phorbol 12-myristate 13-acetate, promoted the cellular processing of hADAM19; however, three calmodulin antagonists, trifluoperazine, W7, and calmidazolium, impaired this cleavage, indicating complex signal pathways may be involved in the processing.     

Modifying the substrate specificity of staphylococcal lipases.

The lipase from Staphylococcus hyicus (SHL) displays a high phospholipase activity whereas the homologous S. aureus lipase (SAL) is not active or hardly active on phospholipid substrates. Previously, it has been shown that elements within the region comprising residues 254-358 are essential for the recognition of phospholipids by SHL. To specifically identify the important residues, nine small clusters of SHL were individually replaced by the corresponding SAL sequence within region 254-358. For cloning convenience, a synthetic gene fragment of SHL was assembled, thereby introducing restriction sites into the SHL gene and optimizing the codon usage. All nine chimeras were well-expressed as active enzymes. Eight chimeras showed lipase and phospholipase activities within a factor of 2 comparable to WT-SHL in standard activity assays. Exchange of the polar SHL region 293-300 by the more hydrophobic SAL region resulted in a 32-fold increased k(cat)/K(m) value for lipase activity and a concomitant 68-fold decrease in k(cat)/K(m) for phospholipase activity. Both changes are due to effects on catalytic turnover as well as on substrate affinity. Subsequently, six point mutants were generated; G293N, E295F, T297P, K298F, I299V, and L300I. Residue E295 appeared to play a minor role whereas K298 was the major determinant for phospholipase activity. The mutation K298F caused a 60-fold decrease in k(cat)/K(m) on the phospholipid substrate due to changes in both k(cat) and K(m). Substitution of F298 by a lysine in SAL resulted in a 4-fold increase in phospholipase activity. Two additional hydrophobic to polar substitutions further increased the phospholipase activity 23-fold compared to WT-SAL.