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1.
Elena Efremenko Ilya Lyagin Yulia Votchitseva Maria Sirotkina Sergey Varfolomeyev 《Biocatalysis and Biotransformation》2013,31(1):103-108
The catalytic and physical–chemical properties of organophosphorus hydrolase (OPH) modified by the addition of an N-terminal dodecahistidine tag (His12-OPH) have been investigated. Introduction of the His12-tag caused a 30- and 74-fold increase in catalytic efficiency of the enzyme with parathion and methyl parathion, respectively, compared to OPH. Concurrently, the His12-OPH had a more alkaline pH-optimum and extended temperature range than OPH and OPH modified with a hexahistidine tag. A study of His12-OPH thermostability showed that the enzyme had a tendency to oligomerise. This resulted in a decrease in the enzymatic activity of His12-OPH at temperatures <50°C, but provided the enzyme with much higher thermostability at temperatures >50°C, compared to OPH. 相似文献
2.
Elena Efremenko Ilya Lyagin Denis Gudkov Sergey Varfolomeyev 《Biocatalysis and Biotransformation》2013,31(2-4):359-364
New biocatalysts were developed using organophosphorus hydrolase (OPH, EC 3.1.8.1) with a polyhistidine tag at the N-terminus of the protein (His6-OPH). The use of His6-OPH together with previously developed approaches for the entrapment of cells into poly(vinyl alcohol) cryogels and covalent immobilization of enzymes into porous fabric materials, impregnated with chemically cross-linked chitosan sulphate gel, enabled dramatic improvement of catalytic characteristics against various organophosphorous compounds (OPCs; Paraoxon, Coumaphos, Methyl parathion, etc.). The polyhistidine tag of OPH was used to create a new immobilized biocatalyst using metal-chelating carriers, such as Ni2+-nitrilotriacetic acid-agarose and Co2+-iminodiacetic acid-polyacrylamide cryogel. The latter biocatalyst had high activity and stability for the continuous hydrolysis of OPCs. 相似文献
3.
In addition to its catalytic domain, phosphoinsositide-dependent protein kinase-1 (PDK1) contains a C-terminal pleckstrin homology (PH) domain, which binds the membrane-bound phosphatidylinositol (3,4,5)-triphosphate [PI(3,4,5)P3] second messenger. Here, we report in vitro kinetic, phosphopeptide mapping, and oligomerization studies that address the role of the PH domain in regulating specific autophosphorylation events, which are required for PDK1 catalytic activation. First, 'inactive' unphosphorylated forms of N-terminal His6 tagged full length (His6-PDK1) and catalytic domain constructs [His6-PDK1(Delta PH)] were generated by treatment with Lambda protein phosphatase (lambda PP). Reconstitution of lambda PP-treated His6-PDK1(Delta PH) catalytic activity required activation loop Ser-241 phosphorylation, which occurred only upon trans-addition of 'active' PDK1 with an apparent bimolecular rate constant of (app)k1(S241) = 374+/-29 M(-1) s(-1). In contrast, full length lambda PP-treated His6-PDK1 catalyzed Ser-241 cis-autophosphorylation with an apparent first-order rate constant of (app)k1(S241) = (5.0+/-1.5) x 10(-4) s(-1) but remained 'inactive'. Reconstitution of lambda PP-treated His(6)-PDK1 catalytic activity occurred only when autophosphorylated in the presence of PI(3,4,5)P3 containing vesicles. PI(3,4,5)P3 binding to the PH domain activated apparent first-order Ser-241 autophosphorylation by 20-fold [(app)k1(S241) = (1.1+/-0.1) x 10(-2) s(-1)] and also promoted biphasic Thr-513 trans-autophosphorylation [(app)k2(T513) = (4.9+/-1.1) x 10(2) M(-1) s(-1) and(app)k3(T513) = (1.5+/-0.2) x 10(3) M(-1) s(-1)]. The results of mutagenesis studies suggest that Thr-513 phosphorylation may cause dissociation of autoinhibitory contacts formed between the contiguous regulatory PH and catalytic kinase domains. 相似文献
4.
An enzyme from the amidohydrolase family from Deinococcus radiodurans (Dr-OPH) with homology to phosphotriesterase has been shown to exhibit activity against both organophosphate (OP) and lactone compounds. We have characterized the physical properties of Dr-OPH and have found it to be a highly thermostable enzyme, remaining active after 3 h of incubation at 60 °C and withstanding incubation at temperatures up to 70 °C. In addition, it can withstand concentrations of at least 200 mg/mL. These properties make Dr-OPH a promising candidate for development in commercial applications. However, compared to the most widely studied OP-degrading enzyme, that from Pseudomonas diminuta, Dr-OPH has low hydrolytic activity against certain OP substrates. Therefore, we sought to improve the OP-degrading activity of Dr-OPH, specifically toward the pesticides ethyl and methyl paraoxon, using structure-based and random approaches. Site-directed mutagenesis, random mutagenesis, and site-saturation mutagenesis were utilized to increase the OP-degrading activity of Dr-OPH. Out of a screen of more than 30,000 potential mutants, a total of 26 mutant enzymes were purified and characterized kinetically. Crystal structures of w.t. Dr-OPH, of Dr-OPH in complex with a product analog, and of 7 mutant enzymes were determined to resolutions between 1.7 and 2.4 Å. Information from these structures directed the design and production of 4 additional mutants for analysis. In total, our mutagenesis efforts improved the catalytic activity of Dr-OPH toward ethyl and methyl paraoxon by 126- and 322-fold and raised the specificity for these two substrates by 557- and 183-fold, respectively. Our work highlights the importance of an iterative approach to mutagenesis, proving that large rate enhancements are achieved when mutations are made in already active mutants. In addition, the relationship between the kinetic parameters and the introduced mutations has allowed us to hypothesize on those factors most important for maintaining the structure and function of the enzyme. 相似文献
5.
Shimizu K Fujino T Ando K Hayakawa M Yasuda H Kikugawa K 《Biochemical and biophysical research communications》2003,304(4):766-771
Oxidized protein hydrolase (OPH) preferentially degrades oxidatively damaged proteins in vitro and is widely distributed in various cells and tissues. The role of OPH in intact cells exposed to oxidative stress was examined. For this purpose, using COS-7, a cell line derived from African green monkey kidney, COS-7-OPH cells that stably overexpressed OPH were established. When COS-7-OPH cells were exposed to oxidative stress induced by H(2)O(2) and paraquat, accumulation of protein carbonyls in the cells was apparently lower than that of parental COS-7 cells, and COS-7-OPH cells were significantly resistant to the oxidative stress compared with parental COS-7 cells. The majority of overexpressed OPH in the cells was found to be located uniformly in cytosol, and its location was not altered by H(2)O(2)-induced oxidative stress. Above results indicate that OPH in intact cells plays a preventive role against oxidative stress and suggest that OPH relieves cells from accumulation of oxidatively damaged proteins. 相似文献
6.
Decontamination of soils with complex pollution using natural strains of microorganisms is a matter of great importance. Here we report that oil-oxidizing bacteria Rhodococcus erythropolis AC-1514D and Rhodococcus ruber AC-1513D can degrade various organophosphorous pesticides (OP). Cell-mediated degradation of five different OP is apparently associated with the presence of N-acylhomoserine lactonase, which is pronouncedly similar (46–50 %) to the well-known enzyme organophosphate hydrolase (OPH), a hydrolysis catalyst for a wide variety of organophosphorous compounds. Additionally, we demonstrated the high lactonase activity of hexahistidine-tagged organophosphate hydrolase (His6-OPH) with respect to various N-acylhomoserine lactones, and we determined the catalytic constants of His6-OPH towards these compounds. These experimental data and theoretical analysis confirmed the hypothesis about the evolutionary proximity of OPH and lactonases. Using Rhodococcus cells, we carried out effective simultaneous biodegradation of pesticide paraoxon (88 mg/kg) and oil hydrocarbon hexadecane (6.3 g/kg) in the soil. Furthermore, the discovered high lactonase activity of His6-OPH offers new possibilities for developing an efficient strategy of combating resistant populations of Gram-negative bacterial cells. 相似文献
7.
The effects of cobalt ion addition and inducer concentration were studied in the fermentation of E. coli BL21 expressing a GFP (green fluorescent protein)-OPH (organophosphorus hydrolase) fusion protein. It was found that cobalt ion addition improved the OPH activity significantly. When 2 mM of CoCl(2) was supplied during the IPTG-induction phase, OPH activity was enhanced approximately 10-fold compared to the case without cobalt or by the addition of cobalt to the cell extracts. Results indicate, therefore, that incorporation of the cobalt during synthesis is needed for enhanced activity. Also, the maximum OPH activity was not linearly related to inducer concentration. A mathematical model was then constructed to simulate these phenomena. Model parameters were determined by constrained least-squares and optimal IPTG and cobalt addition concentrations were obtained, pinpointing the conditions for the maximum productivity. Finally, the GFP fluorescence intensity was found linear to the OPH activity in each fermentation, demonstrating the function of GFP for monitoring its fusion partner's quantity in the bioreactor. 相似文献
8.
Ozer N Bilgi C Hamdi Ogüs I 《The international journal of biochemistry & cell biology》2002,34(3):253-262
Glucose-6-phosphate dehydrogenase (G6PD) catalyses the first step of the pentose phosphate pathway which generates NADPH for anabolic pathways and protection systems in liver. G6PD was purified from dog liver with a specific activity of 130 U x mg(-1) and a yield of 18%. PAGE showed two bands on protein staining; only the slower moving band had G6PD activity. The observation of one band on SDS/PAGE with M(r) of 52.5 kDa suggested the faster moving band on native protein staining was the monomeric form of the enzyme.Dog liver G6PD had a pH optimum of 7.8. The activation energy, activation enthalpy, and Q10, for the enzymatic reaction were calculated to be 8.96, 8.34 kcal x mol(-1), and 1.62, respectively.The enzyme obeyed "Rapid Equilibrium Random Bi Bi" kinetic model with Km values of 122 +/- 18 microM for glucose-6-phosphate (G6P) and 10 +/- 1 microM for NADP. G6P and 2-deoxyglucose-6-phosphate were used with catalytic efficiencies (kcat/Km) of 1.86 x 10(6) and 5.55 x 10(6) M(-1) x s(-1), respectively. The intrinsic Km value for 2-deoxyglucose-6-phosphate was 24 +/- 4mM. Deamino-NADP (d-NADP) could replace NADP as coenzyme. With G6P as cosubstrate, Km d-ANADP was 23 +/- 3mM; Km for G6P remained the same as with NADP as coenzyme (122 +/- 18 microM). The catalytic efficiencies of NADP and d-ANADP (G6P as substrate) were 2.28 x 10(7) and 6.76 x 10(6) M(-1) x s(-1), respectively. Dog liver G6PD was inhibited competitively by NADPH (K(i)=12.0 +/- 7.0 microM). Low K(i) indicates tight enzyme:NADPH binding and the importance of NADPH in the regulation of the pentose phosphate pathway. 相似文献
9.
Metabolic engineering of Pseudomonas putida for the utilization of parathion as a carbon and energy source 总被引:3,自引:0,他引:3
Pseudomonas putida KT2442 was engineered to use the organophosphate pesticide parathion, a compound similar to other organophosphate pesticides and chemical warfare agents, as a source of carbon and energy. The initial step in the engineered degradation pathway was parathion hydrolysis by organophosphate hydrolase (OPH) to p-nitrophenol (PNP) and diethyl thiophosphate, compounds that cannot be metabolized by P. putida KT2442. The gene encoding the native OPH (opd), with and without the secretory leader sequence, was cloned into broad-host-range plasmids under the control of tac and taclac promoters. Expression of opd from the tac promoter resulted in high OPH activity, whereas expression from the taclac promoter resulted in low activity. A plasmid-harboring operons encoding enzymes for p-nitrophenol transformation to beta-ketoadipate was transformed into P. putida allowing the organism to use 0.5 mM PNP as a carbon and energy source. Transformation of P. putida with the plasmids harboring opd and the PNP operons allowed the organism to utilize 0.8 mM parathion as a source of carbon and energy. Degradation studies showed that parathion formed a separate dense, non-aqueous phase liquid phase but was still bioavailable. 相似文献
10.
Takayama K Suye S Kuroda K Ueda M Kitaguchi T Tsuchiyama K Fukuda T Chen W Mulchandani A 《Biotechnology progress》2006,22(4):939-943
The gene encoding organophosphorus hydrolase (OPH) from Flavobacterium species was expressed on the cell surface of Saccharomyces cerevisiae MT8-1 using a glycosylphosphatidylinositol (GPI) anchor linked to the C-terminal region of OPH. Immunofluorescence microscopy confirmed the localization of OPH on the cell surface, and fluorescence intensity measurement of cells revealed that 1.4 x 10(4) molecules of OPH per cell were displayed. Seventy percent of OPH whole-cell activity was detected on the cell surface by protease accessibility assay. The activity of OPH was highly dependent on cell growth conditions. The maximum activity was obtained when cells were grown in a synthetic dextrose medium lacking tryptophan (SD-W) buffered by 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES, 200 mM, pH 7.0) at 20 degrees C, and cobalt chloride was added at 0.1 mM. S. cerevisiae MT8-1 displaying OPH which exhibited a higher activity than Escherichia coli displaying OPH using the ice nucleation protein (INP) anchor. The use of S. cerevisiae MT8-1, which has a "generally regarded as safe (GRAS)" status, as a host for the easy expression of the OPH gene provides a new biocatalyst useful for simultaneous detoxification and detection of organophosphorus pesticides. 相似文献
11.
The kinetics of tissue plasminogen activator (t-PA) and DSPAalpha1-catalyzed plasminogen activation using untreated and TAFIa-treated fibrin degradation products (FDPs), ranging in weight average molecular weight (M(w)) from 0.48 x 10(6) to 4.94 x 10(6) g/mol, were modeled according to the steady-state template model. The FDPs served as effective cofactors for both activators. The intrinsic catalytic efficiencies of both t-PA (17.4 x 10(5) m(-1) s(-1)) and DSPAalpha1 (6.0 x 10(5) m(-1) s(-1)) were independent of FDP M(w). The intrinsic catalytic efficiency of t-PA was 12-fold higher than that measured under identical conditions with intact fibrin as the cofactor. At sub-saturating levels of cofactor and substrate, rates were strongly dependent on FDP M(w) with DSPAalpha1 but not t-PA. Loss of activity with decreasing FDP M(w) correlated with loss of finger-dependent binding of the activators to the FDPs. TAFIa treatment of the FDPs resulted in 90- and 215-fold decreases in the catalytic efficiencies of t-PA (0.20 x 10(5) m(-)(1) s(-1)) and DSPAalpha1 (0.028 x 10(5) m(-1) s(-1)), yielding cofactors that were still 30- and 50-fold better than fibrinogen with t-PA and DSPAalpha1, respectively. Our results show that for both activators the products released during fibrinolysis are very effective cofactors for plasminogen activation, and both t-PA and DSPAalpha1 cofactor activity are strongly down-regulated by TAFIa. 相似文献
12.
A new anchor system based on the ice nucleation protein (InaV) from Pseudomonas syringae INA5 was developed for cell surface display of functional organophosphorus hydrolase (OPH). The activity and stability of cells expressing the truncated InaV (INPNC)-OPH fusions were compared to cells with surface-expressed OPH using two other fusion anchors based on Lpp-OmpA and the truncated InaK protein. Whole cell activity was as much as 5-fold higher using the InaV anchor. Majority of the OPH activity was located on the cell surface as determined by protease accessibility and cell fractionation experiments. The surface localization of OPH was further verified by immunofluorescence microscopy. Constitutive expression of OPH on the surface using the InaV anchor resulted in no cell lysis or growth inhibition, in contrast to the Lpp-OmpA anchor. Suspended cultures also exhibited good stability, retaining almost 100% activity over a period of 3 weeks. Therefore, the InaV anchor system offers an attractive alternative to the currently available surface anchors, providing high-level expression and superior stability. 相似文献
13.
Bønsager BC Praetorius-Ibba M Nielsen PK Svensson B 《Protein expression and purification》2003,32(2):185-193
Barley alpha-amylase/subtilisin inhibitor (BASI) is a beta-trefoil fold protein related to soybean trypsin inhibitor (Kunitz) and inhibits barley alpha-amylase isozyme 2 (AMY2), which is de novo synthesized in the seed during germination. Recombinant BASI was produced in Escherichia coli in an untagged form (untagged rBASI), in two His(6)-tag forms (His(6)-rBASI and His(6)-Xa-rBASI), and in an intein-CBD-tagged form (rBASI (intein)). The yields per liter culture after purification were (i) 25 mgl(-1) His(6)-rBASI; (ii) 6 mgl(-1) rBASI purified after cleavage of His(6)-Xa-rBASI by Factor Xa; (iii) 3 mgl(-1) untagged rBASI; and (iv) 0.2 mgl(-1) rBASI after a chitin-column and autohydrolysis of the rBASI-intein-CBD. In Pichia pastoris, rBASI was secreted at 0.1 mgl(-1). The recombinant BASI forms and natural seed BASI (sBASI) all had an identical isoelectric point of 7.2 and a mass of 19,879 Da, as determined by mass spectrometry. The fold of rBASI from the different preparations was confirmed by circular dichroism spectroscopy and rBASI (intein), His(6)-rBASI, and sBASI inhibited AMY2 catalyzed starch hydrolysis with K(i) of 0.10, 0.06, and 0.09 nM, respectively. Surface plasmon resonance analysis of the formation of AMY2/rBASI (intein) gave k(on)=1.3x10(5)M(-1)s(-1), k(off)=1.4x10(-4)s(-1), and K(D)=1.1 nM, and of the savinase-His(6)-rBASI complex k(on)=21.0x10(4)M(-1)s(-1), k(off)=53.0x10(-4)s(-1), and K(D)=25.0 nM, in agreement with sBASI values. K(i) was 77 and 65 nM for inhibition of savinase activity by His(6)-rBASI and sBASI, respectively. 相似文献
14.
We have characterised the stability, binding and enzymatic properties of three human FK506 binding proteins (FKBP-12) differing only by the length and sequence of their N-terminus. One construct has a short hexa-his tag (6H-FKBP12); the second longer fusion protein (6HL-FKBP12) contains an additional thrombin protease cleavage site; the third has the long fusion tag removed and is essentially native FKBP-12 (cFKBP12). The proteins were purified both under native conditions and also using a refolding protocol. All three natively purified proteins have, within experimental error, the same peptidyl-prolyl isomerase (PPIase) activity (k(cat)/K(m) approximately 1 x 10(6)M(-1)s(-1)), and bind a natural inhibitor, rapamycin, with the same high affinity (K(d) approximately 6 nM). However, refolding of the protein containing the longer tag in vitro results in reduced PPIase activity (the k(cat)/K(m) was reduced from 1 x 10(6)M(-1)s(-1) to 0.81 x 10(6)M(-1)s(-1)) and a 6-fold affinity loss for rapamycin. Addition of both the long and short N-terminal his-tags slows the refolding kinetics of FKBP-12. However, the shorter his-tagged fusion protein regains fully native activity (> or =95%) while the longer regains only approximately 80-85% of native activity. Equilibrium urea denaturation titrations, isothermal titration calorimetry (ITC), analytical gel-filtration, and fluorescence binding data show that this loss of activity is not due to gross misfolding events, but is rather caused by the formation of a stable but subtly misfolded protein that has reduced peptidyl-prolyl isomerase (PPIase) activity and reduced affinity for rapamycin. The difference in behaviour between the in vitro refolded and native forms is due to the dominant role of the cellular chaperone/folding machinery. 相似文献
15.
J S Baumstark 《Comparative biochemistry and physiology. B, Comparative biochemistry》1988,90(1):81-90
1. The principal elastase inhibitor of swine serum, a beta 1-globulin, has been isolated from serum by ammonium sulfate fractionation, DEAE-cellulose column chromatography and chromatofocusing. 2. The purified beta 1-globulin was homogeneous by immunoelectrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Multiple zones (isoinhibitors) were produced on anionic polyacrylamide gels. The mol. wt for the native, elastase-inactivated inhibitor and the beta 1-globulin-elastase complex were respectively, 65,467 and 60,000 and 79,667. The amino acid residue weight was 63,331. 4. The electrophoretic mobilities of the native inhibitor, elastase-inactivated inhibitor and the inhibitor-elastase complex were respectively, -3.4, -3.8 and -2.2 x 10(-5) cm2/V per sec, the isoelectric points were respectively, 4.78-5.28 (major pIs = 5.15, 5.35), 4.63-5.35 (major pI = 5.13) and 6.02-6.2 (major pI = 6.12). 5. The first order dissociation rate constant for the beta 1-globulin-elastase complex (two-fold molar excess of elastase at 37 degrees C) was 1.9 x 10(-3) per sec with complete dissociation in 40.4 min. The dissociation constant for the complex was 1.47 x 10(-7) M. One mol of elastase was bound per mol of the inhibitor. 6. The beta 1-globulin-elastase complex reacts with antibody to either protein moiety. 相似文献
16.
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
17.
A E Senior R S Lee M K al-Shawi J Weber 《Archives of biochemistry and biophysics》1992,297(2):340-344
Nucleotide-depleted Escherichia coli F1 was prepared by the procedure of Wise et al. (1983, Biochem. J. 215, 343-350). This enzyme had high rates of steady-state ATPase and GTPase activity. When "unisite" ATP hydrolysis was measured using an F1/ATP concentration ratio of 10, all of the substoichiometric ATP became bound to the high-affinity catalytic site and none became bound to noncatalytic sites. The association rate constant for ATP binding was 7 x 10(5) M-1 s-1 and the KdATP was 7.9 x 10(-10) M, as compared to values of 3.8 x 10(5) M-1 s-1 and 1.9 x 10(-10) M, respectively, in native (i.e., nucleotide-replete) F1. Rate constants for bound ATP hydrolysis, ATP resynthesis, and P(i) release, and the reaction equilibrium constant, were similar in nucleotide-depleted and native F1. Therefore, we conclude that occupancy of the noncatalytic sites is not required for formation of the high-affinity catalytic site of F1 and has no significant effect on unisite catalysis. In further experiments we looked for the occurrence of inhibitory, catalytic-site-bound MgADP in E. coli F1. Such an entity has been reported for chloroplast and mitochondrial F1. However, our experiments gave no indication for inhibitory MgADP in E. coli F1. 相似文献
18.
Here we report pH dependence of kinetic parameters for the mutarotation of alpha-D-glucose catalyzed by galactose mutarotase (GalM) from Escherichia coli. The values of k(cat) and k(cat)/K(m) for the mutarotation of alpha-D-galactose were found to be 1.84 x 10(4) s(-1) and 4.6 x 10(6) M(-1) s(-1), respectively, at pH 7.0 and 27 degrees C. The corresponding values for alpha-D-glucose were 1.9 x 10(4) s(-1) and 5.0 x 10(5) M(-1) s(-1). Inasmuch as the value of k(cat)/K(m) for the reaction of alpha-D-galactose is 10 times that for alpha-D-glucose, and the diffusional rate constants should be essentially the same for the two sugars, the mutarotation of alpha-D-glucose should not be diffusion controlled. Therefore, pH-rate profiles should not be distorted by diffusion. The k(cat) for the mutarotation of alpha-D-glucose is independent of pH. Therefore, either the enzyme-substrate complexes do not undergo ionization of catalytic groups, or the rate-limiting step is neither mutarotation nor diffusion. The profile of log k(cat)/K(m) versus pH is a distorted bell-shaped curve, with slopes of +1 on the acid side and -2 on the alkaline side. The values of pK(a) are 6.0 and 7.5, and mutarotation depends on the ionization states of three functional groups in the free enzyme, one unprotonated and two protonated. On the acid side, ring opening of alpha-D-glucose limits the rate, and on the alkaline side, ring closure of the open-chain sugar limits the rate. A mutarotation mechanism is presented in which one of the catalytic groups shuttles a proton to and from the endocyclic oxygen and the other two shuttle protons to the anomeric oxygen atoms. In this mechanism, three catalytic groups overcome the problem of nonstereospecificity in mutarotation. The groups are postulated to be His 104, His 175, and Glu 309. Mutations of these residues grossly impair catalytic activity. Variants H104Q- and E309Q-GalM display sufficient activity to allow profiles of log k(cat)/K(m) versus pH to be constructed. Both profiles show breaks on the acid side corresponding to pK(a) values of 5.8 for H104Q and 6.3 for E309Q. Apparently, ring opening of alpha-D-glucose limits the rate at low pHs, but ring closure does not become rate limiting at pHs up to 8.5 in reactions of these variants. 相似文献
19.
Protease nexin II, a platelet-secreted protein containing a Kunitz-type domain, is a potent inhibitor of factor XIa with an inhibition constant of 250-400 pM. The present study examined the protein interactions responsible for this inhibition. The isolated catalytic domain of factor XIa is inhibited by protease nexin II with an inhibition constant of 437 +/- 62 pM, compared to 229 +/- 40 pM for the intact protein. Factor XIa is inhibited by a recombinant Kunitz domain with an inhibition constant of 344 +/- 37 pM versus 422 +/- 33 pM for the catalytic domain. Kinetic rate constants were determined by progress curve analysis. The association rate constants for inhibition of factor XIa by protease nexin II [(3.35 +/- 0.35) x 10(6) M(-1) s(-1)] and catalytic domain [(2.27 +/- 0. 25) x 10(6) M(-1) s(-1)] are nearly identical. The dissociation rate constants are very similar, (9.17 +/- 0.71) x 10(-4) and (7.97 +/- 1.1) x 10(-4) s(-1), respectively. The rate constants for factor XIa and catalytic domain inhibition by recombinant Kunitz domain are also very similar: association constants of (3.19 +/- 0.29) x 10(6) and (3.25 +/- 0.44) x 10(6) M(-1) s(-1), respectively; dissociation constants of (10.73 +/- 0.84) x 10(-4) and (10.36 +/- 1.3) x 10(-4) s(-1). The inhibition constant (K(i)) values calculated from these kinetic parameters are in close agreement with those measured from equilibrium binding experiments. These results suggest that the major interactions required for factor XIa inhibition by protease nexin II are localized to the catalytic domain of factor XIa and the Kunitz domain of protease nexin II. 相似文献
20.
Earlier, we had reported purification of three thiol proteinase inhibitors (TPI-1 of 70 kDa, TPI-3 of 195 kDa and TPI-4 of 497 kDa) from human plasma. In the present study we report that TPI-1 binds to papain in the stoichiometry ratio (E/I) of 1:1 while TPI-3 and TPI-4 bind in the ratio of 1.5:1 and 3.2:1 respectively. The K(m) for papain with BAPNA as substrate and Kcat/K(m) values for TPI-1, TPI-3 and TPI-4 were 2.7 x 10(-6) M, 0.84 nM/sec; 3.2 x 10(-6) M, 0.75 nM/sec; and 3.6 x 10(-6) M, 0.72 nM/sec respectively. The Ki values were found to be 1.48 nM for TPI-1, 0.133 nM for TPI-3 and 0.117 nM for TPI-4. The UV absorption and fluorescence emission spectra study suggest involvement of aromatic residues in the binding process. This study suggests that TPI-4 is the most potent inhibitor of thiol proteinases. 相似文献