共查询到20条相似文献,搜索用时 31 毫秒
1.
Corwin Hansch R.Nelson Smith Arleen Rockoff Daniel F. Calef Priscilla Y.C. Jow James Y. Fukunaga 《Archives of biochemistry and biophysics》1977,183(2):383-392
The parameters Km and kcat were determined for 16 methyl hippurates (CH3OCOCH2NHCOC6H4-X) hydrolyzed by papain. A simple linear relationship is found between log and the hydrophobic substituent constant π. It is found that log kcat is parabolically related to π. The results with papain are compared with results obtained by Hawkins and Williams with the enzyme bromelain. The two enzymes behave in a similar fashion. 相似文献
2.
S-Adenosyl-l-homocysteine hydrolase (EC 3.3.1.1) has been isolated from spinach-beet leaves and purified 100-fold. The enzyme catalyzes both the hydrolysis of S-adenosyl-l-homocysteine to adenosine and l-homocysteine and its synthesis from these compounds. The equilibrium constant for the reaction is 1.8 × 10?6 in relation to hydrolysis. The enzyme shows optimum activity at pH 8.5. Enzyme preparations were stabilized by the addition of bovine serum albumin. The Km for S-adenosylhomocysteine was 41 μm in the hydrolysis reaction and for adenosine, dl-homocysteine, and l-homocysteine it was 13 μm, 2.2 mm, and 1.2 mm, respectively.The enzyme was inhibited by S-adenosylmethionine, homocysteine, and adenine. These inhibitions and the Km values determined are discussed in relation to the regulation of the enzyme in vivo and especially its effect on methylation reactions using S-adenosylmethionine as methyl donor. 相似文献
3.
A comparison of acylation, phosphorylation and binding in related substrates and inhibitors of serum cholinesterase
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1. The Km and catalytic-centre activities for human serum cholinesterase and methyl, ethyl, n-propyl and n-butyl butyrate substrates were determined and compared with the related inhibition constants of a similarly substituted organophosphate inhibitor series based on malaoxon. The results indicated that the catalytic-centre activities approximated to k+2(a), the acylation rate constant, and that Km approximated to the equilibrium binding constant. The inhibition constants measured were Ka, the equilibrium binding constant, and k+2(p), the phosphorylation rate constant. 2. The effects of the alkyl substituents on k+2(p) and k+2(a) were closely parallel, and the decreasing order in each case was: n-butyl; methyl; n-propyl; ethyl. The Taft constants did not follow this order, suggesting that alkyl substituents did not primarily effect acylation or phosphorylation by electron induction. 3. For comparable homologues, the k+2(a) values were on average 435 times the k+2(p) values. The k+2(p) values at 25° and pH7·6 ranged from 6·6min.−1 for the diethyl member to 22·6min.−1 for the di-n-butyl member. 4. The effect of the alkyl substituents on Ka and Km were closely paralleled. The increasing order in each case was: n-butyl; n-propyl; ethyl; methyl. The Ka values were about 100 times less than the comparable Km values. 5. Consideration of the binding energies suggested that only one of the two alkyl groups on the malaoxon homologues bound to the active site. 6. The possibility that malaoxon acted as a substrate as well as an inhibitor for cholinesterase was also investigated, but no evidence of a substrate reaction was found. 相似文献
4.
A kinetic study of oxidative phosphorylation by pea submitochondrial particles gave two Km values for ADP, one low, the other high. The high value probably reflected a damaged site or a population of leaky mitochondria. Only the high affinity site with a low Km for ADP was involved in ATP synthesis. α,β-Methylene ADP was found to be a competitive inhibitor of ATP synthesis. The inorganic phosphate analog, thiophosphate, decreased the apparent Km of ADP while the rate of the reaction remained approximately the same. Adenyl imidodiphosphate, a specific inhibitor of ATP hydrolysis activity, had little effect on oxidative phosphorylation. A slight decrease in the Km of the high affinity binding site for ADP was noted. Aurovertin was found to be a potent inhibitor of oxidative phosphorylation in pea submitochondrial particles. The Km of the high affinity site was increased 10-fold. Also, the inhibition normally exerted by ADP on ATPase activity was severely reduced by aurovertin. In contrast, increasing the concentration of aurovertin only slightly affected the level of inhibition caused by adenyl imidodiphosphate on ATP hydrolysis. 相似文献
5.
Runying Yang 《生物化学与生物物理学报:生物膜》2005,1668(2):248-261
MRP1 transports glutathione-S-conjugated solutes in an ATP-dependent manner by utilizing its two NBDs to bind and hydrolyze ATP. We have found that ATP binding to NBD1 plays a regulatory role whereas ATP hydrolysis at NBD2 plays a dominant role in ATP-dependent LTC4 transport. However, whether ATP hydrolysis at NBD1 is required for the transport was not clear. We now report that ATP hydrolysis at NBD1 may not be essential for transport, but that the dissociation of the NBD1-bound nucleotide facilitates ATP-dependent LTC4 transport. These conclusions are supported by the following results. The substitution of the putative catalytic E1455 with a non-acidic residue in NBD2 greatly decreases the ATPase activity of NBD2 and the ATP-dependent LTC4 transport, indicating that E1455 participates in ATP hydrolysis. The mutation of the corresponding D793 residue in NBD1 to a different acidic residue has little effect on ATP-dependent LTC4 transport. The replacement of D793 with a non-acidic residue, such as D793L or D793N, increases the rate of ATP-dependent LTC4 transport. Along with their higher transport activities, their Michaelis constant Kms (ATP) are also higher than that of wild-type. Coincident with their higher Kms (ATP), their Kds derived from ATP binding are also higher than that of wild-type, implying that the rate of dissociation of the bound nucleotide from the mutated NBD1 is faster than that of wild-type. Therefore, regardless of whether the bound ATP at NBD1 is hydrolyzed or not, the release of the bound nucleotide from NBD1 may bring the molecule back to its original conformation and facilitate the protein to start a new cycle of ATP-dependent solute transport. 相似文献
6.
We report for the first time an analysis of the ATPase activity of human DNA topoisomerase (topo) IIβ. We show that topo IIβ is a DNA-dependent ATPase that appears to fit Michaelis–Menten kinetics. The ATPase activity is stimulated 44-fold by DNA. The kcat for ATP hydrolysis by human DNA topo IIβ in the presence of DNA is 2.25 s–1. We have characterised a topo IIβ derivative which carries a mutation in the ATPase domain (S165R). S165R reduced the kcat for ATP hydrolysis by 7-fold, to 0.32 s–1, while not significantly altering the apparent Km. The specificity constant for the interaction between ATP and topo IIβ (kcat/Kmapp) showed a 90% reduction for βS165R. The DNA binding affinity and ATP-independent DNA cleavage activity of the enzyme are unaffected by this mutation. However, the strand passage activity is reduced by 80%, presumably due to reduced ATP hydrolysis. The mutant enzyme is unable to complement ts yeast topo II in vivo. We have used computer modelling to predict the arrangement of key residues at the ATPase active site of topo IIβ. Ser165 is predicted to lie very close to the bound nucleotide, and the S165R mutation could thus influence both ATP binding and ADP dissociation. 相似文献
7.
The 70-kD family of heat shock proteins (Hsp70s) is involved in a number of seemingly disparate cellular functions, including folding of nascent proteins, breakup of misfolded protein aggregates, and translocation of proteins across membranes. They act through the binding and release of substrate proteins, accompanied by hydrolysis of ATP. Chloroplast stromal Hsp70 plays a crucial role in the import of proteins into plastids. Mutations of an ATP binding domain Thr were previously reported to result in an increase in the Km for ATP and a decrease in the enzyme’s kcat. To ask which chloroplast stromal chaperone, Hsp70 or Hsp93, both of which are ATPases, dominates the energetics of the motor responsible for protein import, we made transgenic moss (Physcomitrella patens) harboring the Km-altering mutation in the essential stromal Hsp70-2 and measured the effect on the amount of ATP required for protein import into chloroplasts. Here, we report that increasing the Km for ATP hydrolysis of Hsp70 translated into an increased Km for ATP usage by chloroplasts for protein import. This thus directly demonstrates that the ATP-derived energy long known to be required for chloroplast protein import is delivered via the Hsp70 chaperones and that the chaperone’s ATPase activity dominates the energetics of the reaction. 相似文献
8.
《BBA》1987,893(2):197-207
The apparent Michaelis constant (Km) for ADP has been measured under various conditions of steady-state photophosphorylation in isolated thylakoid membranes. In addition, the steady-state ΔpH has been simultaneously estimated from the fluorescence quenching of 9-aminoacridine. The following results were obtained. (1) The standard procedure for estimating Km, by increasing the concentration of ADP, progressively lowered the steady-state ΔpH, thereby introducing an uncontrolled system variable into the Km analysis. This has the effect of lowering the apparent Km measured. (2) Lowering the light intensity lowered the observed Km, and addition of uncouplers increased the observed Km. The ability of uncouplers to increase Km was enhanced at lowered light intensities. In contrast, the effect of lowered light intensity on the observed Km was diminished and then reversed under progressively more uncoupled conditions. (3) The addition of energy-transfer inhibitors caused an increase in the observed Km for ADP. (4) All of the observations are qualitatively predicted by a mathematical model based on simple delocalised chemiosmotic energy coupling and Michaelis-Menten kinetics for the chloroplast ATPase with respect to ADP. It is concluded that the complex behaviour of the apparent Km for ADP under different conditions arises because ΔpH is an uncontrolled variable during the Km analysis and that the results are entirely consistent with a model of delocalised chemiosmotic energy coupling. 相似文献
9.
Transport and metabolism of 1'-fluorosucrose, a sucrose analog not subject to invertase hydrolysis 总被引:1,自引:1,他引:0
The novel sucrose derivative 1′-fluorosucrose (α-d-glucopyranosyl-β- d-1-deoxy-1-fluorofructofuranoside) was synthesized in order to help define mechanisms of sucrose entry into plant cells. Replacement of the 1′-hydroxyl by fluorine very greatly reduces invertase hydrolysis of the derivative (hydrolysis at 10 millimolar 1′-fluorosucrose is less than 2% that of sucrose) but does not reduce recognition, binding, or transport of 1′-fluorosucrose by a sucrose carrier. Transport characteristics of 1′-fluorosucrose were studied in three different tissues. The derivative is transported by the sucrose carrier in the plasmalemma of developing soybean cotyledon protoplasts with a higher affinity than sucrose (Km 1′-fluorosucrose 0.9 millimolar, Km sucrose 2.0 millimolar). 1′-Fluorosucrose is a competitive inhibitor of sucrose uptake with an apparent Ki also of 0.9 millimolar, while the Ki of sucrose competition of 1′-fluorosucrose uptake was 2.0 millimolar. Thus, both sugars are recognized at the same binding site in the plasmalemma. Both sucrose and 1′-fluorosucrose show very similar patterns of phloem translocation from an abraded leaf surface through the petiole indicating that recognition of 1′-fluorosucrose by sucrose carriers involved in phloem loading is likely as well. 相似文献
10.
Effects of lipid removal on the molecular size and kinetic properties of bovine plasma arylesterase 总被引:1,自引:1,他引:0
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A purified arylesterase preparation from bovine plasma was characterized to the extent that it has a partial specific volume of 0.91ml/g and an apparent z-average molecular weight of 440000. The relatively large magnitude of the former reflects the presence of phospholipids, cholesterol, triglycerides and β-carotene, the last-named being responsible for the pronounced yellow colour of the preparation. Removal of the lipid material is accompanied by a decrease in the apparent z-average molecular weight to 120000, the size of the smallest species detected by high-speed sedimentation equilibrium being in the vicinity of 70000 daltons: denaturation of the lipid-free preparation with 6m-guanidine hydrochloride caused essentially complete breakdown into subunits of this size. In kinetic studies on the enzyme the maximal velocity for the hydrolysis of phenyl acetate was found to increase by 60% on addition of 1 mm-Ca2+, with the Km showing a concomitant decrease from 6.6 to 2.1 mm. Removal of lipid had no detectable effect on Vmax. or Km in either the presence or the absence of Ca2+. It is concluded that the bovine plasma arylesterase preparation is either a lipoprotein or an enzyme–lipoprotein complex with properties very similar to those of the α1-lipoprotein or high-density lipoprotein (HDL2) fraction of serum. 相似文献
11.
A low molecular weight acid phosphatase was purified to homogeneity from chicken heart with a specific activity of 42 U/mg
and a recovery of about 1%. Nearly 800 fold purification was achieved. The molecular weight was estimated to be 18 kDa by
SDS-polyacrylamide gel electrophoresis. Para-nitrophenyl phosphate, phenyl phosphate and flavin mononucleotide were efficiently hydrolysed by the enzyme and found to
be good substrates. Fluoride and tartrate had no inhibitory effect while phosphate, vanadate and molybdate strongly inhibited
the enzyme. The acid phosphatase was stimulated in the presence of glycerol, ethylene glycol, methanol, ethanol and acetone,
which reflected the phosphotransferase activity. When phosphate acceptors such as ethylene glycol concentrations were increased,
the ratio of phosphate transfer to hydrolysis was also increased, demonstrating the presence of a transphosphorylation reaction
where an acceptor can compete with water in the rate limiting step involving hydrolysis of a covalent phospho enzyme intermediate.
Partition experiments carried out with two substrates, para-nitrophenyl phosphate and phenyl phosphate, revealed a constant product ratio of 1.7 for phosphotransfer to ethylene glycol
versus hydrolysis, strongly supporting the existence of common covalent phospho enzyme intermediate. A constant ratio of K
cat/K
m, 4.3×104, found at different ethylene glycol concentrations, also supported the idea that the rate limiting step was the hydrolysis
of the phospho enzyme intermediate. 相似文献
12.
A comparison was made of graphical and subtractive methods for the determination of the dissociation constant of a complex between ferredoxin:NADP reductase and NADP. The subtractive method gave Kd values near 10 μm which are consistent with recently determined values for Km,NADP in assays of NADP photoreduction by chloroplast membranes. The graphical method gave values which were considerably higher. The difference between the two methods is due to the failure of the graphical method to correct for the amount of each component present in the complex at the low NADP/ flavoprotein ratios necessary for binding studies. A second NADP binding site of much lower affinity (Kd approx 1 mm) was also detected. 相似文献
13.
The kinetics of the hydrolysis of 3-(2-furylacryloyl)-glycycl-l-leucine amide by thermolysin has been reinvestigated. It was found that the Km for the enzyme substrate interaction is 2.5 × 10?3m at pH 7.2. This Km is an order of magnitude less than what has been previously assumed to be the Km for the enzyme-substrate interaction. The normally recommended assay has 1–3 × 10?3m substrate and is based on the assumption that the substrate concentration is much less than the Km. Our data indicate that this assumption appears to be invalid. The hydrolysis of 3-(2-furylacryloyl)-glycyl-l-leucine amide results in a maximum decrease in absorbance at 322 nm. The change in absorbance is nearly 10-fold greater at 322 nm than the change in absorbance at 345 nm where the hydrolysis has been customarily followed. By following the hydrolysis of the substrate at 10?4m at 322 nm it is possible to work under conditions where the substrate concentration is much less than the Km. 相似文献
14.
Inhibition of the Fermentation of Propionate to Methane by Hydrogen, Acetate, and Propionate 总被引:5,自引:3,他引:2
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Satoshi Fukuzaki Naomichi Nishio Manabu Shobayashi Shiro Nagai 《Applied microbiology》1990,56(3):719-723
Inhibition of the fermentation of propionate to methane and carbon dioxide by hydrogen, acetate, and propionate was analyzed with a mesophilic propionate-acclimatized sludge that consisted of numerous flocs (size, 150 to 300 μm). The acclimatized sludge could convert propionate to methane and carbon dioxide stoichiometrically without accumulating hydrogen and acetate in a propionate-minimal medium. Inhibition of propionate utilization by propionate could be analyzed by a second-order substrate inhibition model (shown below) given that the substrate saturation constant, Ks, was 15.9 μM; the substrate inhibition constant, Ki, was 0.79 mM; and the maximum specific rate of propionate utilization, qm, was 2.15 mmol/g of mixed-liquor volatile suspended solids (MLVSS) per day: qs = qmS/[Ks + S + (S2/Ki)], where qs is the specific rate of propionate utilization and S is the initial concentration of undissociated propionic acid. For inhibition by hydrogen and acetate to propionate utilization, a noncompetitive product inhibition model was used: qs = qm/[1 + (P/Kp)n], where P is the initial concentration of hydrogen or undissociated acetic acid and Kp is the inhibition constant. Kinetic analysis gave, for hydrogen inhibition, Kp(H2) = 0.11 atm (= 11.1 kPa, 71.5 μM), qm = 2.40 mmol/g of MLVSS per day, and n = 1.51 and, for acetate inhibition, Kp(HAc) = 48.6 μM, qm = 1.85 mmol/g of MLVSS per day, and n = 0.96. It could be concluded that the increase in undissociated propionic acid concentration was a key factor in inhibition of propionate utilization and that hydrogen and acetate cooperatively inhibited propionate degradation, suggesting that hydrogenotrophic and acetoclastic methanogens might play an important role in enhancing propionate degradation to methane and carbon dioxide. 相似文献
15.
The effect of metal ions on human activated Factor X (Factor Xa) hydrolysis of the chromogenic substrate benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S2222) was studied utilizing initial rate enzyme kinetics. The divalent metal ions Ca2+, Mn2+, and Mg2+ enhanced Factor Xa amidolytic activity with Km values of 30 μm, 20 μm, and 1.4 mm, respectively. Na+ activation of Factor Xa amidolytic activity was also found. The Km for Na+ activation was 0.31 m. Both the divalent metal ions and Na+ increased the affinity of Factor Xa for S2222 and had no effect on the maximal velocity of the reaction. Other monovalent cations were unable to activate Factor Xa. However, K+ was a competitive inhibitor of the Na+ activation (Ki = 0.14 m). Lanthanide ions inhibited Factor Xa amidolytic activity. Gd3+ inhibition of Factor Xa hydrolysis of S2222 was noncompetitive and had a Ki of 3 μm. The lanthanide ion inhibition could not be reversed by Ca2+ even when Ca2+ was present in a 1000-fold excess over its Km indicating nonidentity of the Factor Xa lanthanide and Ca2+ binding sites. It is concluded that the Factor Xa Ca2+ binding sites have characteristics different from those previously described for the Factor X molecule and that Mg2+, Na+, and K+ may be physiological regulators of Factor Xa activity. 相似文献
16.
Takayoshi Tagami Keitaro Yamashita Masayuki Okuyama Haruhide Mori Min Yao Atsuo Kimura 《The Journal of biological chemistry》2015,290(3):1796-1803
The α-glucosidase from sugar beet (SBG) is an exo-type glycosidase. The enzyme has a pocket-shaped active site, but efficiently hydrolyzes longer maltooligosaccharides and soluble starch due to lower Km and higher kcat/Km for such substrates. To obtain structural insights into the mechanism governing its unique substrate specificity, a series of acarviosyl-maltooligosaccharides was employed for steady-state kinetic and structural analyses. The acarviosyl-maltooligosaccharides have a longer maltooligosaccharide moiety compared with the maltose moiety of acarbose, which is known to be the transition state analog of α-glycosidases. The clear correlation obtained between log Ki of the acarviosyl-maltooligosaccharides and log(Km/kcat) for hydrolysis of maltooligosaccharides suggests that the acarviosyl-maltooligosaccharides are transition state mimics. The crystal structure of the enzyme bound with acarviosyl-maltohexaose reveals that substrate binding at a distance from the active site is maintained largely by van der Waals interactions, with the four glucose residues at the reducing terminus of acarviosyl-maltohexaose retaining a left-handed single-helical conformation, as also observed in cycloamyloses and single helical V-amyloses. The kinetic behavior and structural features suggest that the subsite structure suitable for the stable conformation of amylose lowers the Km for long-chain substrates, which in turn is responsible for higher specificity of the longer substrates. 相似文献
17.
Yakov M. Milgrom 《BBA》2010,1797(10):1768-1774
The effect of inorganic phosphate (Pi) on uni-site ATP binding and hydrolysis by the nucleotide-depleted F1-ATPase from beef heart mitochondria (ndMF1) has been investigated. It is shown for the first time that Pi decreases the apparent rate constant of uni-site ATP binding by ndMF1 3-fold with the Kd of 0.38 ± 0.14 mM. During uni-site ATP hydrolysis, Pi also shifts equilibrium between bound ATP and ADP + Pi in the direction of ATP synthesis with the Kd of 0.17 ± 0.03 mM. However, 10 mM Pi does not significantly affect ATP binding during multi-site catalysis. 相似文献
18.
《Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology》1994,1204(2):164-168
The role of carboxyl group in the catalytic action of xylanase (Mr 35 000) from an alkalothermophilic Bacillus sp. was delineated through iinetic and chemical modification studies using Woodward's Reagent K. The kinetics of inactivation indicated that one carboxyl residue was essential for the xylanase activity with a second order rate constant of 3300 M−1 min−1. The spectrophotometric analysis at 340 nm revealed that the inhibition was correlated with modification of 24 carboxyl residues. In the presence of protecting ligand, modification of one carboxyl group was prevented. The pH profile showed apparent pK values of 5.2 and 6.4 for the free enzyme and 4.9 and 6.9 for enzyme-substrate complex. The pH dependence of inactivation was consistent with the modification of carboxyl group. The kinetic analysis of the modified enzyme showed similar Km and lower kcat values than the native enzyme indicating that catalytic hydrolysis and not the substrate binding was affected by chemical modification. The chemical modification of xylanase from alkalothermophilic Bacillus revealed the presence of tryptophans in the active site (Dehspande, V, Hinge, J. and Rao, M. (1990) Biochim. Biophys. Acta 1041, 172–177). This finding and present studies demonstrated the experimental evidence for the participation of carboxyl as well as tryptophan groups as essential residues of xylanase from alkalothermophilic Bacillus sp. 相似文献
19.
Lars Örning Sven Hammarström 《Biochemical and biophysical research communications》1982,106(4):1304-1309
γ-Glutamyl transpeptidase (EC 2.3.2.2) converts leukotriene C to leukotriene D by removal of a glutamyl residue. The Michaelis constant for leukotriene C4 hydrolysis was found to be 5.6 μM. Under the same conditions the Km value for hydrolysis of reduced glutathione was 5.7 μM. This suggests that leukotriene C4 and glutathione may be competing substrates for γ-glutamyl transpeptidase under physiological conditions. The apparent KI for inhibition of leukotriene C4 hydrolysis by equimolar amounts of L-serine and sodium borate was 0.8 mM. 相似文献
20.
F.M.A.H. Schuurmans Stekhoven H.G.P. Swarts J.J.H.H.M. De Pont S.L. Bonting 《生物化学与生物物理学报:生物膜》1983,732(3):607-619
(1) The Mg2+-induced low-affinity nucleotide binding by (Na+ + K+)-ATPase has been further investigated. Both heat treatment (50–65°C) and treatment with N-ethylmaleimide reduce the binding capacity irreversibly without altering the Kd value. The rate constant of inactivation is about one-third of that for the high-affinity site and for the (Na+ + K+)-ATPase activity. (2) Thermodynamic parameters (ΔH° and ΔS°) for the apparent affinity in the ATPase reaction (Km ATP) and for the true affinity in the binding of AdoPP[NH]P (Kd and Ki) differ greatly in sign and magnitude, indicating that one or more reaction steps following binding significantly contribute to the Km value, which thus is smaller than the Kd value. (3) Ouabain does not affect the capacity of low-affinity nucleotide binding, but only increases the Kd value to an extent depending on the nucleotide used. GTP and CTP appear to be most sensitive, ATP and ADP intermediately sensitive and AdoPP[NH]P and least sensitive to ouabain. Ouabain reduces the high-affinity nucleotide binding capacity without affecting the Kd value. (4) The nucleotide specificity of low-affinity binding site is the same for binding (competition with AdoPP[NH]P) and for the ATPase activity (competition with ATP): AdoPP[NH]P > ATP > ADP > AMP. (5) The low-affinity nucleotide binding capacity is preserved in the ouabain-stabilized phosphorylated state, and the Kd value is not increased more than by ouabain alone. (6) It is inferred that the low-affinity site is Iocated on the enzyme, more specifically its α-subunit, and not on the surrounding phospholipids. It is situated outside the phosphorylation centre. The possible functional role of the low-affinity binding is discussed. 相似文献