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1.
N Kadohama C Yarborough D Zhou S Chen Y Osawa 《The Journal of steroid biochemistry and molecular biology》1992,43(7):693-701
Mutant forms of aromatase cytochrome P-450 bearing modifications of amino acid residues Pro308 and Asp309 and expressed in transfected Chinese hamster ovary cells were subjected to kinetic analysis and inhibition studies. The Km for androstenedione for expressed wild type (11.0 +/- 0.3 nM SEM, n = 3) increased 4-, 25- and 31-fold for mutants Pro308Phe, Asp309Asn and Asp309Ala, respectively. There were significant differences in sensitivity among wild type and mutants to highly selective inhibitors of estrogen biosynthesis. 4-Hydroxyandrostenedione (4-OHA) a strong inhibitor of wild type aromatase activity (IC50 = 21 nM and Ki = 10 nM), was even more effective against mutant Pro308Phe (IC50 = 13 nM and Ki = 2.8 nM), but inhibition of mutants Asp309Asn and Asp309Ala was considerably less (IC50 = 345 and 330 nM and Ki = 55 and 79 nM, respectively). Expressed wild type aromatase and Pro308Phe aromatase were strongly inhibited by CGS 16949A (IC50 = 4.0 and 4.6 nM, respectively) whereas mutants Asp309Asn and Asp309Ala were markedly less sensitive (IC50 = 140 and 150 nM, respectively). CGS 18320B produced similar inhibition. Kinetic analyses produced Ki = 0.4 nM for CGS 16949A inhibition of wild type versus 1.1, 37 and 58 nM, respectively, against Pro308Phe, Asp309Asn and Asp309Ala. The results demonstrate significant changes in function resulting from single amino acid modifications of the aromatase enzyme. Our data indicate that mutation in Asp309 creates a major distortion in the substrate binding site, rendering the enzyme much less efficient for androstenedione aromatization. The substitution of Pro308 with Phe produces weaker affinity for androstenedione in the substrate pocket, but this alteration favors 4-OHA binding. Similarly, mutant Pro308Phe exhibits a slightly greater sensitivity to inhibition by CGS 18320B than does the wild type. These results indicate that residues Pro308 and Asp309 play critical roles in determining substrate specificity and catalytic capability in aromatase. 相似文献
2.
Nogami K Freas J Manithody C Wakabayashi H Rezaie AR Fay PJ 《The Journal of biological chemistry》2004,279(32):33104-33113
The 337-372 sequence of the factor VIIIa A1 subunit contains interactive sites for both zymogen factor X and the active enzyme, factor Xa. Solid phase binding studies indicated that factor Xa possessed a >20-fold higher affinity for the isolated A1 subunit of factor VIIIa compared with factor X. Heparin completely inhibited zero-length cross-linking of the 337-372 peptide to factor Xa but not to factor X. In the presence of calcium, factor Xa showed greater affinity for heparin than factor X. Studies using factor Xa mutants in which heparin-binding exosite residues were individually replaced by Ala showed that the R240A mutant was defective in recognition of the Lys36 cleavage site, generating the A137-372 intermediate with approximately 20% the catalytic efficiency of wild type. This defect likely resulted from an approximately 4-fold increase in Km for the A1 substrate because kcat values for the wild type and mutant were equivalent. Cleavage of the A1-A2 domain junction by factor Xa R240A was not blocked by the 337-372 peptide. Studies using mutant factor VIII where clustered acidic residues in the 337-372 segment were replaced by Ala showed that a factor VIIIa D361A/D362A/D363A mutant possessed a approximately 1.6-fold increase in Km for factor X compared with wild type. However, similar Km values were observed for recombinant factor X and R240A substrates. These results indicate that the binding regions of factor X and factor Xa for A1 domain overlap and that both utilize acidic residues 361-363. Furthermore, factor Xa but not factor X interacts with high affinity at this site via residues contained within the heparin-binding exosite of the proteinase. 相似文献
3.
Oh EJ Lee YJ Chol JJ Seo MS Lee MS Kim GA Kwon ST 《Journal of microbiology and biotechnology》2008,18(2):287-294
Three amino acid residues (His119, Glu164, and Glu338) in the active site of Thermus caldophilus GK24 beta- glycosidase (Tca beta-glycosidase), a family 1 glycosyl hydrolase, were mutated by site-directed mutagenesis. To verify the key catalytic residues, Glu164 and Glu338 were changed to Gly and Gln, respectively. The E164G mutation resulted in drastic reductions of both beta-galactosidase and beta-glucosidase activities, and the E338Q mutation caused complete loss of activity, confirming that the two residues are essential for the reaction process of glycosidic linkage hydrolysis. To investigate the role of His119 in substrate binding and enzyme activity, the residue was substituted with Gly. The H119G mutant showed 53-fold reduced activity on 5 mM p-nitrophenyl beta-Dgalactopyranoside, when compared with the wild type; however, both the wild-type and mutant enzymes showed similar activity on 5 mM p-nitrophenyl beta-D-glucopyranoside at 75degreeC. Kinetic analysis with p-nitrophenyl beta-D-galactopyranoside revealed that the kcat value of the H119G mutant was 76.3-fold lower than that of the wild type, but the Km of the mutant was 15.3-fold higher than that of the wild type owing to the much lower affinity of the mutant. Thus, the catalytic efficiency (kcat/Km) of the mutant decreased to 0.08% to that of the wild type. The kcat value of the H119G mutant for p-nitrophenyl beta- D-glucopyranoside was 5.1-fold higher than that of the wild type, but the catalytic efficiency of the mutant was 2.5% of that of the wild type. The H119G mutation gave rise to changes in optima pH (from 5.5-6.5 to 5.5) and temperature (from 90 degrees C to 80-85 degrees C). This difference of temperature optima originated in the decrease of H119G's thermostability. These results indicate that His119 is a crucial residue in beta- galactosidase and beta-glucosidase activities and also influences the enzyme's substrate binding affinity and thermostability. 相似文献
4.
J D Clausen D B McIntosh D G Woolley J P Andersen 《The Journal of biological chemistry》2001,276(38):35741-35750
Mutants in which Thr-353 of the Ca(2+)-ATPase of sarcoplasmic reticulum had been replaced with alanine, serine, glutamine, cysteine, valine, aspartate, or tyrosine were analyzed functionally. All the mutations severely affected MgATP binding, whereas ATP binding was close to normal in the alanine, serine, glutamine, and valine mutants. In the serine and valine mutants, the maximum rate of phosphorylation from MgATP was 8- and 600-fold lower, respectively, compared with wild type. Replacement of Mg(2+) with Mn(2+) led to a 1.5-fold enhancement of the maximum phosphorylation rate in the valine mutant and a 5-fold reduction in the wild type. The turnover of the phosphoenzyme formed from MgATP was slowed 1-2 orders of magnitude relative to wild type in the alanine, serine, and valine mutants, but was close to normal in the aspartate and cysteine mutants. Only the serine mutant formed a phosphoenzyme in the backward reaction with P(i), and the hydrolysis of this intermediate was greatly enhanced. Analysis of the functional changes in the mutants in the light of the recent high resolution structure of the Ca(2+)-ATPase crystallized without the MgATP substrate suggests that, in the native activated state of the enzyme, the side chain hydroxyl of Thr-353 participates in important interactions with nucleotide and phosphate, possibly in catalysis, whereas the main chain carbonyl of Thr-353, but not the side chain, may coordinate the catalytic Mg(2+). 相似文献
5.
Sequence alignment predicts that His(309) of pig heart NADP-dependent isocitrate dehydrogenase is equivalent to His(339) of the Escherichia coli enzyme, which interacts with the coenzyme in the crystal structure [Hurley et al. (1991) Biochemistry 30, 8671-8688], and porcine His(315) and His(319) are close to that site. The mutant porcine enzymes H309Q, H309F, H315Q, and H319Q were prepared by site-directed mutagenesis, expressed in E. coli, and purified. The H319Q mutant has K(m) values for NADP, isocitrate, and Mn(2+) similar to those of wild-type enzyme, and V(max) = 20.1, as compared to 37.8 micromol of NADPH min(-1) (mg of protein)(-1) for wild type. Thus, His(319) is not involved in coenzyme binding and has a minimal effect on catalysis. In contrast, H315Q exhibits a K(m) for NADP 40 times that of wild type and V(max) = 16.2 units/mg of protein, with K(m) values for isocitrate and Mn(2+) similar to those of wild type. These results implicate His(315) in the region of the NADP site. Replacement of His(309) by Q or F yields enzyme with no detectable activity. The His(309) mutants bind NADPH poorly, under conditions in which wild type and H319Q bind 1.0 mol of NADPH/mol of subunit, indicating that His(309) is important for the binding of coenzyme. The His(309) mutants bind isocitrate stoichiometrically, as do wild-type and the other mutant enzymes. However, as distinguished from the wild-type enzyme, the His(309) mutants are not oxidatively cleaved by metal isocitrate, implying that the metal ion is not bound normally. Since circular dichroism spectra are similar for wild type, H315Q, and H319Q, these amino acid substitutions do not cause major conformational changes. In contrast, replacement of His(309) results in detectable change in the enzyme's CD spectrum and therefore in its secondary structure. We propose that His(309) plays a significant role in the binding of coenzyme, contributes to the proper coordination of divalent metal ion in the presence of isocitrate, and maintains the normal conformation of the enzyme. 相似文献
6.
Arginine residues in the D2 polypeptide may stabilize bicarbonate binding in photosystem II of Synechocystis sp. PCC 总被引:1,自引:0,他引:1
Bicarbonate (HCO3-) causes a significant and reversible stimulation of anion-inhibited electron flow in photosystem II of higher plants and cyanobacteria. To test if selected arginine (Arg) residues are involved in the binding of HCO3-, we utilized oligonucleotide-directed mutagenesis to construct Synechocystis sp. PCC 6803 mutants carrying mutations in Arg residues in the D2 protein. Measurements of oxygen evolution showed that the D2 mutants R233Q (arginine-233----glutamine) and R251S (arginine-251----serine) were 10-fold more sensitive to formate than the wild type. The formate concentration giving half-maximal inhibition of the steady-state oxygen evolution rate was 48 mM, 4.5 mM and 4 mM for the wild type, R233Q and R251S, respectively. Measurements of oxygen evolution in single-turnover flashes confirm that the mutants are more sensitive to formate than the wild type. Measurements of chlorophyll a fluorescence decay kinetics after the second saturating actinic flash indicated that, after formate treatment, the halftime of QA- oxidation was decreased by approximately a factor of 2, 4 and 6 in the wild type, R251S and R233Q, respectively. The recombination rate between QA- and S2 was approx. 2-fold slower in R251S and R233Q than in the wild type. In the presence of 100 mM sodium formate, reactivation of the Hill reaction by bicarbonate showed that the wild type had an apparent Km for bicarbonate of 0.5 mM, while the Km values for R233Q and R251S were 1.4 and 1.5 mM, respectively. We suggest that Arg-233 and Arg-251 in the D2 polypeptide contribute to stabilization of HCO3- binding in Photosystem II. 相似文献
7.
S-Adenosylmethionine (AdoMet) synthetase catalyzes the biosynthesis of AdoMet in a unique enzymatic reaction. Initially the sulfur of methionine displaces the intact tripolyphosphate chain (PPP(i)) from ATP, and subsequently PPP(i) is hydrolyzed to PP(i) and P(i) before product release. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site contains four aspartate residues. Aspartate residues Asp-16* and Asp-271 individually provide the sole protein ligand to one of the two required Mg(2+) ions (* denotes a residue from a second subunit); aspartates Asp-118 and Asp-238* are proposed to interact with methionine. Each aspartate has been changed to an uncharged asparagine, and the metal binding residues were also changed to alanine, to assess the roles of charge and ligation ability on catalytic efficiency. The resultant enzyme variants all structurally resemble the wild type enzyme as indicated by circular dichroism spectra and are tetramers. However, all have k(cat) reductions of approximately 10(3)-fold in AdoMet synthesis, whereas the MgATP and methionine K(m) values change by less than 3- and 8-fold, respectively. In the partial reaction of PPP(i) hydrolysis, mutants of the Mg(2+) binding residues have >700-fold reduced catalytic efficiency (k(cat)/K(m)), whereas the D118N and D238*N mutants are impaired less than 35-fold. The catalytic efficiency for PPP(i) hydrolysis by Mg(2+) site mutants is improved by AdoMet, like the wild type enzyme. In contrast AdoMet reduces the catalytic efficiency for PPP(i) hydrolysis by the D118N and D238*N mutants, indicating that the events involved in AdoMet activation are hindered in these methionyl binding site mutants. Ca(2+) uniquely activates the D271A mutant enzyme to 15% of the level of Mg(2+), in contrast to the approximately 1% Ca(2+) activation of the wild type enzyme. This indicates that the Asp-271 side chain size is a discriminator between the activating ability of Ca(2+) and the smaller Mg(2+). 相似文献
8.
Jacques SL Nieman C Bareich D Broadhead G Kinach R Honek JF Wright GD 《Biochimica et biophysica acta》2001,1544(1-2):28-41
Fungal homoserine dehydrogenase (HSD) is required for the biosynthesis of threonine, isoleucine and methionine from aspartic acid, and is a target for antifungal agents. HSD from the yeast Saccharomyces cerevisiae was overproduced in Escherichia coli and 25 mg of soluble dimeric enzyme was purified per liter of cell culture in two steps. HSD efficiently reduces aspartate semialdehyde to homoserine (Hse) using either NADH or NADPH with kcat/Km in the order of 10(6-7) M(-1) x s(-1) at pH 7.5. The rate constant of the reverse direction (Hse oxidation) was also significant at pH 9.0 (kcat/Km approximately 10(4-5) M(-1) x s(-1)) but was minimal at pH 7.5. Chemical modification of HSD with diethyl pyrocarbonate (DEPC) resulted in a loss of activity that could be obviated by the presence of substrates. UV difference spectra revealed an increase in absorbance at 240 nm for DEPC-modified HSD consistent with the modification of two histidines (His) per subunit. Amino acid sequence alignment of HSD illustrated the conservation of two His residues among HSDs. These residues, His79 and His309, were substituted to alanine (Ala) using site directed mutagenesis. HSD H79A had similar steady state kinetics to wild type, while kcat/Km for HSD H309A decreased by almost two orders of magnitude. The recent determination of the X-ray structure of HSD revealed that His309 is located at the dimer interface [B. DeLaBarre, P.R. Thompson, G.D. Wright, A.M. Berghuis, Nat. Struct. Biol. 7 (2000) 238-244]. The His309Ala mutant enzyme was found in very high molecular weight complexes rather than the expected dimer by analytical gel filtration chromatography analysis. Thus the invariant His309 plays a structural rather than catalytic role in these enzymes. 相似文献
9.
An unexpected role for the active site base in cofactor orientation and flexibility in the copper amine oxidase from Hansenula polymorpha. 总被引:1,自引:0,他引:1
The role of the active site aspartate base in the aminotransferase mechanism of the copper amine oxidase from the yeast Hansenula polymorpha has been probed by site-directed mutagenesis. The D319E mutant catalyzes the oxidation of methylamine and phenethylamine, but not that of benzylamine. kcat/Km for methylamine is found to be 80-fold reduced compared to that of the wild type. Viscosogen and substrate and solvent deuteration have no effect on this parameter for D319E, which is suggestive of limitation of kcat/Km by a conformational change. This conformational change is proposed to be the movement of the cofactor into a productive orientation upon the binding of substrate. In the absence of substrate, a flipped cofactor orientation is likely, on the basis of resonance Raman evidence that the C5 carbonyl of the cofactor is less solvent accessible than the C3 hydrogen. kcat for D319E methylamine oxidase is reduced 200-fold compared to that of the wild type and is unaffected by substrate deuteration, but displays a substantial solvent isotope effect. A 428 nm absorbance is evident under conditions of saturating methylamine and oxygen with D319E. The D319N mutant is observed to produce a similar absorbance at 430 nm when treated with ammonia despite the fact that this mutant has no amine oxidase activity. Resonance Raman spectroscopy indicates the formation of a covalent ammonia adduct and identifies it as the deprotonated iminoquinone. In contrast, when the D319E mutant is reacted with ammonia, it gives predominantly a 340-350 nm species. This absorbance is ascribed to a localization of the cofactor oxyanion induced by binding of the cation at the active site and not to covalent adduct formation. Resonance Raman spectroscopic examination of the steady state species of D319E methylamine oxidation, in combination with the kinetic data, indicates that the 428 nm species is the deprotonated iminoquinone produced upon reoxidation of the reduced cofactor. A model is proposed in which a central role of the active site base is to position the free cofactor and several enzyme intermediates for optimal activity. 相似文献
10.
The homology model of the wild type alginate lyase (AlyVI) marine bacterium Vibrio sp. protein, was built using the crystal structure of the Family 7 alginate lyase from Sphingomonas sp. A1. To rationalize the observed structure-affinity relationships of aliginate lyase alyVI with its (GGG) substrate, molecular docking, MD imulations and binding free energy calculations followed by site-directed mutagenesis and alyVI activity assays were carried out. Per-residue decomposition of the (GGG) binding energy revealed that the most important contributions were from polar and charged residues, such as Asn138, Arg143, Asn217, and Lys308, while van der Waals interactions were responsible for binding with the catalytic His200 and Tyr312 residues. The mutants H200A, K308A, Y312A, Y312F, and W165A were found to be inactive or almost inactive. However, the catalytic efficiency (k(cat)/K(m)) of the double mutant L224V/D226G increased by two-fold compared to the wild type enzyme. This first structural model with its substrate binding mode and the agreement with experimental results provide a suitable base for the future rational design of new mutated alyVI structures with improved catalytic activity. 相似文献
11.
Matsunaga I Ueda A Sumimoto T Ichihara K Ayata M Ogura H 《Archives of biochemistry and biophysics》2001,394(1):45-53
CYP152A1 is an unusual, peroxygenase enzyme that catalyzes the beta- or alpha-hydroxylation of fatty acids by efficiently introducing an oxygen atom from H2O2 to the fatty acid. To clarify the mechanistic roles of amino acid residues in this enzyme, we have used site-directed mutagenesis of residues in the putative distal helix and measured the spectroscopic and enzymatic properties of the mutant proteins. Initially, we carried out Lys-scanning mutagenesis of amino acids in this region to determine residues of CYP152A1 that might have a mechanistic role. Among the Lys mutants, only P243K gave an absorption spectrum characteristic of a nitrogenous ligand-bound form of a ferric P450. Further investigation of the Pro243 site revealed that a P243H mutant also exhibited a nitrogen-bound form, but that the mutants P243A or P243S did not. On the hydroxylation of myristic acid by the Lys mutants, we observed a large decrease in activity for R242K and A246K. We therefore examined other mutants at amino acid positions 242 and 246. At position 246, an A246K mutant showed a roughly 19-fold lower affinity for myristic acid than the wild type. Replacing Ala246 with Ser decreased the catalytic activity, but did not affect affinity for the substrate. An A246V mutant showed slightly reduced activity and moderately reduced affinity. At position 242, an R242A showed about a fivefold lower affinity than the wild type for myristic acid. The Km values for H2O2 increased and Vmax values decreased in the order of wild type, R242K, and R242A when H2O2 was used; furthermore, Vmax/Km was greatly reduced in R242A compared with the wild type. If cumene hydroperoxide was used instead of H2O2, however, the Km values were not affected much by these substitutions. Together, our results suggest that in CYP152A1 the side chain of Pro243 is located close to the iron at the distal side of a heme molecule; the fatty acid substrate may be positioned near to Ala246 in the catalytic pocket, although Ala246 does not participate in hydrophobic interactions with the substrate; and that Arg242 is a critical residue for substrate binding and H2O2-specific catalysis. 相似文献
12.
Altered metabolism of thrombospondin by Chinese hamster ovary cells defective in glycosaminoglycan synthesis 总被引:17,自引:0,他引:17
J E Murphy-Ullrich L G Westrick J D Esko D F Mosher 《The Journal of biological chemistry》1988,263(13):6400-6406
We examined the ability of Chinese hamster ovary (CHO) cell mutants defective in glycosaminoglycan synthesis to metabolize 125I-labeled thrombospondin (TSP). Wild type CHO cells bound and degraded 125I-TSP with kinetics similar to those reported for endothelial cells. Both binding and degradation were saturable (half-saturation at 20 micrograms/ml). When the concentration of labeled TSP was 1-5 micrograms/ml, mutant 745, defective in xylosyltransferase, and mutant 761, defective in galactosyltransferase I, bound and degraded 6- to 16-fold less TSP than wild type; mutant 803, which specifically lacks heparan sulfate chains, bound and degraded 5-fold less TSP than wild type; and mutant 677, which lacks heparan sulfate and has increased levels of chondroitin sulfate, bound and degraded 2-fold less TSP than wild type. Binding and degradation of TSP by the mutants were not saturable at TSP concentrations up to 100 micrograms/ml. Bound TSP was localized by immunofluorescence to punctate structures on wild type and, to a lesser extent, 677 cells. Heparitinase pretreatment of wild type cells caused a 2- to 3-fold decrease in binding and degradation, whereas chondroitinase pretreatment had no effect. Chondroitinase pretreatment of the 677 mutant (deficient heparan sulfate and excess chondroitin sulfate) caused a 2-fold decrease in binding and an 8-fold decrease in turnover, whereas heparitinase pretreatment had no effect. Treatment of wild type cells with both heparitinase and chondroitinase resulted in a 6- to 8-fold decrease in binding and turnover. These results indicate that cell surface proteoglycans mediate metabolism of TSP by CHO cells and that the primary effectors of TSP metabolism are heparan sulfate proteoglycans. 相似文献
13.
K Shirabe T Yubisui T Nishino M Takeshita 《The Journal of biological chemistry》1991,266(12):7531-7536
Human NADH-cytochrome b5 reductase (EC 1.6.2.2) contains 4 cyteine residues (Cys-203, -273, -283, and -297). Cys-283 was previously proposed to be involved in NADH binding by chemical modification (Hackett, C. S., Novoa, W. B., Ozols, J., and Strittmatter, P. (1986) J. Biol. Chem. 261, 9854-9857). In the present study the role of cysteines in the enzyme was probed by replacing these residues by Ser, Ala, or Gly employing site-directed mutagenesis and chemical modification. Four mutants, in which 1 of the 4 Cys residues was replaced by Ser, retained comparable kcat and Km values to those of the wild type. All of these mutants were as sensitive as the wild type to treatment with SH modifiers, while a double mutant, C273S/C283S was resistant. Since inhibition by SH modifiers was protected by NADH, Cys-273 and Cys-283 were implicated to be close to the NADH-binding site. C273A and C273A/C283A mutants showed approximately one-fifth of the enzyme-FAD reduction rate of the wild type as revealed by steady-state kinetics and by stopped-flow analysis. Anaerobic titration has shown that reduction and re-oxidation processes including formation of the red semiquinone of these mutants were not significantly altered from those of the wild type. From these results it was concluded that none of the Cys residues of the enzyme are essential in the catalytic reaction, but Cys-273 conserved among the enzymes homologous to NADH-cytochrome b5 reductase homologous to NADH-cytochrome b5 reductase plays role(s) in facilitating the reaction. A difference spectrum with a peak at 317 nm, which was formerly considered to be derived from the interaction between NAD+ and Cys-283 of the reduced enzyme, appeared upon binding of NAD+ not only to the reduced wild type enzyme but also to the C273A/C283A mutant in which both of the Cys residues close to the NADH-binding site were replaced. 相似文献
14.
A highly conserved amino acid sequence 442GDASE446 in the ATP binding pocket of rat Na/K-ATPase was mutated, and the resulting proteins, G442A, G442P, D443A, S445A, and E446A, were expressed in HeLa cells to investigate the effect of individual ligands on Na/K-ATPase. The apparent Km for the high and low affinity ATP effects was estimated by ATP concentration dependence for the formation of the Na-dependent phosphoenzyme (Kmh) and Na/K-ATPase activity (Kml). The apparent Km for p-nitrophenylphosphate (pNPP) for K-dependent-pNPPase (KmP) and its inhibition by ATP (Ki,0.5) and the apparent Km for Mg2+, Na+, K+, and vanadate in Na/K-ATPase were also estimated. For all the mutants, the value for ATP was approximately 2-10-fold larger than that of the wild type. While the turnover number for Na/K-ATPase activity were unaffected or reduced by 20 approximately 50% in mutants G442(A/P) and D443A. Although both affinities for ATP effects were reduced as a result of the mutations, the ratio, Kml Kmh, for each mutant was 1.3 approximately 3.7, indicating that these mutations had a greater impact on the low affinity ATP effect than on the high affinity effect. Each KmP value with the turnover number suggests that these mutations favor the binding of pNPP over that of ATP. These data and others indicate that the sequence 442GDASE446 in the ATP binding pocket is an important motif that it is involved in both the high and low affinity ATP effects rather than in free Mg2+, Na+, and K+ effects. 相似文献
15.
Waldow A Schmidt B Dierks T von Bülow R von Figura K 《The Journal of biological chemistry》1999,274(18):12284-12288
Arylsulfatase A belongs to the sulfatase family whose members carry a Calpha-formylglycine that is post-translationally generated by oxidation of a conserved cysteine or serine residue. The formylglycine acts as an aldehyde hydrate with two geminal hydroxyls being involved in catalysis of sulfate ester cleavage. In arylsulfatase A and N-acetylgalactosamine 4-sulfatase this formylglycine was found to form the active site together with a divalent cation and a number of polar residues, tightly interconnected by a net of hydrogen bonds. Most of these putative active site residues are highly conserved among the eukaryotic and prokaryotic members of the sulfatase family. To analyze their function in binding and cleaving sulfate esters, we substituted a total of nine putative active site residues of human ASA by alanine (Asp29, Asp30, Asp281, Asn282, His125, His229, Lys123, Lys302, and Ser150). In addition the Mg2+-complexing residues (Asp29, Asp30, Asp281, and Asn282) were substituted conservatively by either asparagine or aspartate. In all mutants Vmax was decreased to 1-26% of wild type activity. The Km was more than 10-fold increased in K123A and K302A and up to 5-fold in the other mutants. In all mutants the pH optimum was increased from 4.5 by 0.2-0.8 units. These results indicate that each of the nine residues examined is critical for catalytic activity, Lys123 and Lys302 by binding the substrate and the others by direct (His125 and Asp281) or indirect participation in catalysis. The shift in the pH optimum is explained by two deprotonation steps that have been proposed for sulfate ester cleavage. 相似文献
16.
A cDNA encoding mature porcine heart aconitase was over-expressed in Escherichia coli under the control of a phage T7 promoter. Recombinant aconitase purified from E. coli was identical to the enzyme from pig and beef heart in size, [3Fe-4S] and [4Fe-4S] cluster structure and enzymatic activity. Nine amino acid residues in close proximity to the Fe-S cluster and bound substrate (Lauble, H., Kennedy, M.C., Beinert, H., and Stout, C.D. (1992) Biochemistry, in press) were replaced by site-directed mutagenesis. Fe-S cluster environment as indicated by the EPR spectrum, tight binding of substrate, and enzymatic activity were compared for the mutant and wild type enzymes. Significant perturbations were detected for all of the mutant enzymes. Replacements for Asp100, His101, Asp165, Arg580, and Ser642 result in a 10(3)-10(5)-fold drop in activity, which suggests that these residues are involved in critical aspects of the reaction. Arg580 appears to be a key residue for substrate binding, as shown by a 30-fold increased Km and loss of tight substrate binding. Results of mutagenesis support the interpretation of the x-ray model, namely that Asp100 and His101 form an ion pair for elimination of the substrate hydroxyl and Ser642 may function as a general base for proton abstraction from citrate or isocitrate in the dehydration step and protonation of cis-aconitate in the hydration step. Asp165 appears to play a critical role in the interaction of Fea with substrate. 相似文献
17.
Based on the crystal structure of human topoisomerase I, we hypothesized that hydrogen bonding between the side chain of the highly conserved His(632) and one of the nonbridging oxygens of the scissile phosphate contributes to catalysis by stabilizing the transition state. This hypothesis has been tested by examining the effects of changing His(632) to glutamine, asparagine, alanine, and tryptophan. The change to glutamine reduced both the relaxation activity and single-turnover cleavage activity by approximately 100-fold, whereas the same change at three other conserved histidines (positions 222, 367, and 406) had no significant effect on the relaxation activity. The properties of the mutant protein containing asparagine instead of histidine at position 632 were similar to those of the glutamine mutant, whereas mutations to alanine or tryptophan reduced the activity by approximately 4 orders of magnitude. The reduction in activity for the mutants was not due to alterations in substrate binding affinities or changes in the cleavage specificities of the proteins. The above results for the glutamine mutation in conjunction with the similar effects of pH on the wild type and the H632Q mutant enzyme rule out the possibility that His(632) acts as a general acid to protonate the leaving 5'-oxygen during the cleavage reaction. Taken together, these data strongly support the hypothesis that the only role for His(632) is to stabilize the pentavalent transition state through hydrogen bonding to one of the nonbridging oxygens. 相似文献
18.
Adenosylcobalamin-dependent methylmalonyl-CoA mutase catalyzes the interconversion of methylmalonyl-CoA and succinyl-CoA via radical intermediates generated by substrate-induced homolysis of the coenzyme carbon-cobalt bond. From the structure of methylmalonyl-CoA mutase it is evident that the deeply buried active site is completely shielded from solvent with only a few polar contacts made between the protein and the substrate. Site-directed mutants of amino acid His244, a residue close to the inferred site of radical chemistry, were engineered to investigate its role in catalysis. Two mutants, His244Ala and His244Gln, were characterized using kinetic and spectroscopic techniques. These results confirmed that His244 is not an essential residue. However, compared with that of the wild type, k(cat) was lowered by 10(2)- and 10(3)-fold for the His244Gln and His244Ala mutants, respectively, while the K(m) for succinyl-CoA was essentially unchanged in both cases. The primary kinetic tritium isotope effect (k(H)/k(T)) for the His244Gln mutant was 1.5 +/- 0.3, and tritium partitioning was now found to be dependent on the substrate used to initiate the reaction, indicating that the rearrangement of the substrate radical to the product radical was extremely slow. The His244Ala mutant underwent inactivation under aerobic conditions at a rate between 1 and 10% of the initial rate of turnover. The crystal structure of the His244Ala mutant, determined at 2.6 A resolution, indicated that the mutant enzyme is unaltered except for a cavity in the active site which is occupied by an ordered water molecule. Molecular oxygen reaching this cavity may lead directly to inactivation. These results indicate that His244 assists directly in the unusual carbon skeleton rearrangement and that alterations in this residue substantially lower the protection of reactive radical intermediates during catalysis. 相似文献
19.
Dihydrofolate reductase (EC 1.5.1.3) is a key enzyme in the folate biosynthetic pathway. Information regarding key residues in the dihydrofolate-binding site of Mycobacterium avium dihydrofolate reductase is lacking. On the basis of previous information, Asp31 and Leu32 were selected as residues that are potentially important in interactions with dihydrofolate and antifolates (e.g. trimethoprim), respectively. Asp31 and Leu32 were modified by site-directed mutagenesis, giving the mutants D31A, D31E, D31Q, D31N and D31L, and L32A, L32F and L32D. Mutated proteins were expressed in Escherichia coli BL21(DE3)pLysS and purified using His-Bind resin; functionality was assessed in comparison with the recombinant wild type by a standard enzyme assay, and growth complementation and kinetic parameters were evaluated. All Asp31 substitutions affected enzyme function; D31E, D31Q and D31N reduced activity by 80-90%, and D31A and D31L by > 90%. All D31 mutants had modified kinetics, ranging from three-fold (D31N) to 283-fold (D31L) increases in K(m) for dihydrofolate, and 12-fold (D31N) to 223 077-fold (D31L) decreases in k(cat)/K(m). Of the Leu32 substitutions, only L32D caused reduced enzyme activity (67%) and kinetic differences from the wild type (seven-fold increase in K(m); 21-fold decrease in k(cat)/K(m)). Only minor variations in the K(m) for NADPH were observed for all substitutions. Whereas the L32F mutant retained similar trimethoprim affinity as the wild type, the L32A mutation resulted in a 12-fold decrease in affinity and the L32D mutation resulted in a seven-fold increase in affinity for trimethoprim. These findings support the hypotheses that Asp31 plays a functional role in binding of the substrate and Leu32 plays a functional role in binding of trimethoprim. 相似文献
20.
D. Tan M. J. Barber G. C. Ferreira 《Protein science : a publication of the Protein Society》1998,7(5):1208-1213
5-Aminolevulinate synthase (EC 2.3.1.37) is the first enzyme in the heme biosynthesis in nonplant eukaryotes and some prokaryotes. It functions as a homodimer and requires pyridoxal 5'-phosphate as an essential cofactor. Tyr-121 is a conserved residue in all known sequences of 5-aminolevulinate synthases. Further, it corresponds to Tyr-70 of Escherichia coli aspartate aminotransferase, which has been shown to interact with the cofactor and prevent the dissociation of the cofactor from the enzyme. To test whether Tyr-121 is involved in cofactor binding in murine erythroid 5-aminolevulinate synthase, Tyr-121 of murine erythroid 5-aminolevulinate synthase was substituted by Phe and His using site-directed mutagenesis. The Y121F mutant retained 36% of the wild-type activity and the Km value for substrate glycine increased 34-fold, while the activity of the Y121H mutant decreased to 5% of the wild-type activity and the Km value for glycine increased fivefold. The pKa1 values in the pH-activity profiles of the wild-type and mutant enzymes were 6.41, 6.54, and 6.65 for wild-type, Y121F, and Y121H, respectively. The UV-visible and CD spectra of Y121F and Y121H mutants were similar to those of the wild-type with the exception of an absorption maximum shift (420 --> 395 nm) for the Y121F mutant in the visible spectrum region, suggesting that the cofactor binds the Y121F mutant enzyme in a more unrestrained manner. Y121F and Y121H mutant enzymes also exhibited lower affinity than the wild-type for the cofactor, reflected in the Kd values for pyridoxal 5'-phosphate (26.5, 6.75, and 1.78 microM for Y121F, Y121H, and the wild-type, respectively). Further, Y121F and Y121H proved less thermostable than the wild type. Taken together, these findings indicate that Tyr-121 plays a critical role in cofactor binding of murine erythroid 5-aminolevulinate synthase. 相似文献