共查询到20条相似文献,搜索用时 15 毫秒
1.
Di Stasio E Lancellotti S Peyvandi F Palla R Mannucci PM De Cristofaro R 《Biophysical journal》2008,95(5):2450-2461
The zinc-protease a disintegrin-like and metalloprotease with thrombospondin type I repeats (ADAMTS13) cleaves the Tyr1605-Met1606 peptide bond of von Willebrand factor (VWF), avoiding the accumulation of ultra large VWF multimers. Hydrolysis by ADAMTS13 of a VWF analog (Asp1596-Arg1668 peptide, fluorescence energy transfer substrate [FRETS]-VWF73) was investigated by a fluorescence quenching method (FRETS method) from 15°C to 45°C and pH values from 4.5 to 10.5. The catalysis was influenced by two ionizable groups, whose pKa values were equal to 6.41 ± 0.08 (ionization enthalpy = 32.6 ± 1.7 kJ/mol) and 4 ± 0.1 (ionization enthalpy = 3.8 ± 0.4 kJ/mol), whereas these values were equal to 6 ± 0.1 and 4.1 ± 0.1, respectively, in Co2+-substituted ADAMTS13. The catalytic process of FRETS-VWF73 hydrolysis showed negative activation entropy (−144 kJ/mol), suggesting that the transition state becomes more ordered than the ground state of the reactants. The kcat/Km values were not linearly correlated with temperature, as expression of change of the kinetic “stickiness” of the substrate. The Met1606-Arg1668 peptide product acted as hyperbolic mixed-type inhibitor of FRETS-VWF73 hydrolysis. Asp1653, Glu1655, Glu1660, Asp1663, together with the hydrophilic side chain of Thr1656 were shown to form a “hot spot” in the VWF A2 sequence, which drives the molecular recognition and allosteric regulation of binding to ADAMTS13. The interaction of the Met1606-Arg1668 region of VWF with ADAMTS13 involves basic residues of the protease and is thus progressively inhibited at pH values >8.50. A molecular model of the FRETS-VWF73 showed that the substrate can fit into the active site only if ADAMTS13 assumes a C-like shape and, interacting with the acidic 1653-1668 region of VWF, properly orients the Tyr1605-Met1606 peptide bond for the cleavage by the zinc-aquo complex in the active site. 相似文献
2.
Mechanistic studies of peptidyl prolyl cis-trans isomerase: evidence for catalysis by distortion 总被引:9,自引:0,他引:9
Cyclophilin, the cytosolic binding protein for the immunosuppressive drug cyclosporin A, has recently been shown to be identical with peptidyl prolyl cis-trans isomerase [Fischer, G., Wittmann-Liebold, B., Lang, K., Kiefhaber, T., & Schmid, F.X. (1989) Nature 337, 476; Takahashi, N., Hayano, T., & Suzuki, M. (1989) Nature 337, 473]. To provide a mechanistic framework for studies of the interaction of cyclophilin with cyclosporin, we investigated the mechanism of the PPI-catalyzed cis to trans isomerization of Suc-Ala-Xaa-cis-Pro-Phe-pNA (Xaa = Ala, Gly). Our mechanistic studies of peptidyl prolyl cis-trans isomerase include the determination of steady-state kinetic parameters, pH and temperature dependencies, and solvent and secondary deuterium isotope effects. The results of these experiments support a mechanism involving catalysis by distortion in which the enzyme uses free energy released from favorable, noncovalent interactions with the substrate to stabilize a transition state that is characterized by partial rotation about the C-N amide bond. 相似文献
3.
Mechanistic studies of cyclohexanone monooxygenase: chemical properties of intermediates involved in catalysis 总被引:3,自引:0,他引:3
Cyclohexanone monooxygenase (CHMO), a bacterial flavoenzyme, carries out an oxygen insertion reaction on cyclohexanone to form a seven-membered cyclic product, epsilon-caprolactone. The reaction catalyzed involves the four-electron reduction of O2 at the expense of a two-electron oxidation of NADPH and a two-electron oxidation of cyclohexanone to form epsilon-caprolactone. Previous studies suggested the participation of either a flavin C4a-hydroperoxide or a flavin C4a-peroxide intermediate during the enzymatic catalysis [Ryerson, C. C., Ballou, D. P., and Walsh, C. (1982) Biochemistry 21, 2644-2655]. However, there was no kinetic or spectral evidence to distinguish between these two possibilities. In the present work we used double-mixing stopped-flow techniques to show that the C4a-flavin-oxygen adduct, which is formed rapidly from the reaction of oxygen with reduced enzyme in the presence of NADP, can exist in two states. When the reaction is carried out at pH 7.2, the first intermediate is a flavin C4a-peroxide with maximum absorbance at 366 nm; this intermediate becomes protonated at about 3 s(-1) to form what is believed to be the flavin C4a-hydroperoxide with maximum absorbance at 383 nm. These two intermediates can be interconverted by altering the pH, with a pK(a) of 8.4. Thus, at pH 9.0 the flavin C4a-peroxide persists mainly in the deprotonated form. Further kinetic studies also demonstrated that only the flavin C4a-peroxide intermediate could oxygenate the substrate, cyclohexanone. The requirement in catalysis of the deprotonated flavin C4a-peroxide, a nucleophile, is consistent with a Baeyer-Villiger rearrangement mechanism for the enzymatic oxygenation of cyclohexanone. In the course of these studies, the Kd for cyclohexanone to the C4a-peroxyflavin form of CHMO was determined to be approximately 1 microM. The rate-determining step in catalysis was shown to be the release of NADP from the oxidized enzyme. 相似文献
4.
Recent work demonstrating the ability of spliceosomes purified after the second catalytic step of splicing to efficiently reverse both steps of the reaction provides answers to several unresolved questions regarding the splicing reaction, and raises many more. 相似文献
5.
6.
《Journal of enzyme inhibition and medicinal chemistry》2013,28(3):370-376
N-Acetylgalactosamine kinase (GALK2) is a small molecule kinase from the GHMP family which phosphorylates N-acetylgalactosamine at the expense of ATP. Recombinant GALK2 expressed in, and purified from, Escherichia coli was shown to be active with the following kinetic parameters: Michaelis constant for ATP, 14?±?3?μM; Michaelis constant for N-acetylgalactosamine, 40?±?14?μM; and turnover number, 1.0?±?0.1?s?1. The combination of substrate inhibition by N-acetylgalactosamine and α-methylgalactopyranoside acting as an uncompetitive inhibitor with respect to ATP suggested that the enzyme has an ordered ternary complex mechanism in which ATP is the first substrate to bind. The effects of pH on the kinetic parameters provided evidence for ionizable residues playing a role in substrate binding and catalysis. These results are discussed in the context of the mechanisms of the GHMP kinases. 相似文献
7.
Kovári J Imre T Szabó P Vértessy BG 《Nucleosides, nucleotides & nucleic acids》2004,23(8-9):1475-1479
The essential enzyme dUTPase is responsible for preventive DNA repair via exclusion of uracil. Lack or inhibition of the enzyme induces thymine-less cell death in cells performing active DNA synthesis, serving therefore as an important chemotherapeutic target. In the present work, employing differential circular dichroism spectroscopy, we show that D. mel. dUTPase, a recently described eukaryotic model, has a similar affinity of binding towards alpha,beta-imino-dUTP as compared to the prokaryotic E. coli enzyme. However, in contrast to the prokaryotic dUTPase, the nucleotide exerts significant protection against tryptic digestion at a specific tryptic site 20 A far from the active site in the fly enzyme. This result indicates that binding of the nucleotide in the active site induces an allosteric conformational change within the central threefold channel of the homotrimer exclusively in the eukaryotic enzyme. Nucleotide binding induced allosterism in the D. mel. dUTPase, but not in the E. coli enzyme, might be associated with the altered hydropathy of subunit interfaces in these two proteins. 相似文献
8.
Francis W. Ballardie Brian Capon Murray W. Cuthbert W. M. Dearie 《Bioorganic chemistry》1977,6(4):483-509
The preparation of some aryl β-glycosides of β-1, 4-linked oligosaccharides of (GlcNAc)n, n = 2, 3, 4, is described. These compounds were tested as substrates for lysozyme from hens' egg white. The best of them, (GlcNAc)4-3,4-DNP, had a value of kcat/Km which was about one-nintieth that for the hydrolysis of (GlcNAc)6. The pH dependence of kcat and kcat/Km for the hydrolysis of (GlcNAc)4-3,4-DNP was similar to that for (GlcNAc)6. (GlcNAc)4-3,4-DNP was also a substrate for human lysozyme and lysozyme from ducks' egg white (II and III). An impure sample of (GlcNAc)2F was prepared and this was hydrolyzed much more rapidly than (GlcNAc)2-2,4-DNP by lysozyme. Compounds of type (GlcNAc)n−1(XylNAc)Ar, where n = 2, 3, 4, were prepared and found not to be substrates for lysozyme. In the presence of (GlcNAc)4 or (GlcNAc)5, lysozyme-induced hydrolyses of (GlcNAc)-3,4-DNP and (Glc)-3,4-DNP were observed but not of (XylNAc)-3,4-DNP, (6-deoxy-GlcNAc)-3,4-DNP, (6-F-GlcNAc)-3,4-DNP, and (6-Cl-GlcNAc)-3,4-DNP. The significance of these results is discussed. 相似文献
9.
In vitro selection has allowed the isolation of many new ribozymes that are able to catalyze an ever-widening array of chemical transformations. Mechanistic studies on these selected ribozymes have provided valuable insight into the methods that RNA can invoke to overcome different catalytic tasks. We focus on the methods employed in these mechanistic studies using the acyl-transferase family of selected ribozymes as well-studied reference systems. Chemical and biochemical techniques have been used in tandem in order to draw conclusions on the various modes of catalysis employed by the different family members. In turn, this type of mechanistic information may provide a means for the redesign and optimization of existing ribozymes or the basis for new selection systems for more powerful RNA catalysts. 相似文献
10.
《Journal of Biomedical Engineering》1985,7(3):183-195
A knowledge of the chemical composition of blood, urine and other body fluids is a daily requirement for departments of biochemistry. Electronic circuitry and computers to process the data are readily available, but satisfactory transducers to convert chemical composition into electrical signals are frequently the weakest link in the chain of measurement. This review is concerned with one group of transducers: ion-selective electrodes.Since the commercial success of the calcium and fluoride ions-selective electrodes in the mid-sixties, a range of other electrodes has become available. Their use has already conferred considerable benefits upon medicine, e.g. rapid, low-cost, multiple assay of major blood components; diagnostic surveys of chloride sweat levels relating to cystic fibrosis; and monitoring blood fluorides during and after halothane administration. Their adoption for indirectly sensing enzymes and associated substrates is particularly noteworthy. Recent advances in electronics, coupled with flow injection schemes based on ion-selective electrodes, have facilitated the management of hundreds of samples daily.However, developments in the mechanistic knowledge of these sensors have not matched the increase in their application, although definite progress can be reported; for example with regard to the origin of the potential signals induced by ion activities in solution. Numerous techniques have been devised to unravel mechanistic problems, among which radioisotope tracer and impedance measurements may be cited as especially valuable.Selectivity performance, particularly in complex biological media, and undesirable features such as protein poisoning, need further research. Organic chemists are now better placed to synthesize new designs of acyclic and cyclic molecules as mobile site, ion-selective, sensor materials which, with appropriate mediator solvents, provide improved sensor cocktails. This design feature is well illustrated by the continuing quest for a lithium ion-selective electrode compatible with the high levels of sodium interference in blood. 相似文献
11.
Faller LD 《Archives of biochemistry and biophysics》2008,476(1):12-21
Sodium pump was the first ion pump discovered. A member of the family of active transporters that catalyze adenosine 5′-triphosphate hydrolysis by forming a phosphorylated enzyme intermediate, sodium pump couples the energy released to unequal countertransport of sodium and potassium ions. The ion gradient generated by the pump is important for a variety of secondary physiological processes ranging from metabolite transport to electrical excitation of nerve and muscle. Selected experiments relating structure to function are reviewed. 相似文献
12.
Mechanistic studies on the high pressure neurological syndrome 总被引:3,自引:0,他引:3
F Bowser-Riley 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》1984,304(1118):31-41
The high pressure neurological syndrome (h.p.n.s.) constitutes a major barrier to deep sea exploration by man. Although the signs and symptoms of h.p.n.s. are well documented in both man and experimental animals, the underlying mechanisms remain to be elucidated. Physiological and pharmacological evidence will be presented that confirms that the principal sites of action of pressure are within the central nervous system (c.n.s.). Results from physiological studies not only indicate that there are separate sites of action of pressure within the c.n.s., which mediate the different components of h.p.n.s., but also the response to pressure may be controlled by descending inhibitory pathways. Pharmacological studies support this view and suggest that h.p.n.s. involves a failure of central inhibition. 相似文献
13.
The reaction mechanism of Azospirillum brasilense glutamate synthase has been investigated by several approaches. 15N nuclear magnetic resonance studies demonstrate that the amide nitrogen of glutamine is reductively transferred to 2-oxoglutarate in an irreversible manner with no release of the transferred ammonia group into the medium. Identical results were obtained using thio-NADPH and acetylpyridine-NADPH, which are shown to be less efficient substrates of the enzyme than NADPH. Similarly, no exchange of the ammonia group being transferred with exogenous ammonium ion was observed during catalysis. The glutamate formed as the product of the iminoglutarate reduction was determined to be in the L configuration. The enzyme was also found to catalyze, under anaerobic conditions, the exchange of the 4proS H of NADPH with solvent both in the absence and in the presence of 2-oxoglutarate and glutamine. The reductive half-reaction is therefore a reversible segment of the overall irreversible amidotransferase reaction. 15N NMR studies also showed that the enzyme does not catalyze glutamate dehydrogenase/oxidase reactions or any observable glutaminase activity under neutral (pH 7.5) conditions. Glutaminase activity was also not observable with the reduced enzyme alone or in the presence of D-glutamate (a competitive inhibitor of glutamate synthase with respect to 2-oxoglutarate, with a Ki of about 11 microM) or with the oxidized enzyme in the presence of 2-oxoglutarate, D-glutamate, or NADP+. These data confirm species-dependent differences of A. brasilense glutamate synthase with respect to the enzyme from other sources. 相似文献
14.
Fluorescence polarization has been used to study the kinetics of the combination of thrombin with antithrombin and its catalysis by the polysaccharide heparin. The heparin-catalysed combination of thrombin and antithrombin is saturable with respect to both thrombin and antithrombin. The rate-determining step of the reaction is approximately 1.7 s-1. The kinetics observed can be explained by proposing that the catalyst of the reaction is not heparin alone but a complex of heparin and antithrombin (bound at the high-affinity site). The temperature dependence of the heparin-catalysed reaction is indistinguishable from that of the uncatalysed reaction. This coincidence is consistent with the rate-limiting step being the same in both cases. 相似文献
15.
To probe the mechanism of stromelysin (SLN)-catalyzed peptide hydrolysis, we determined the pH dependence of kc/Km and solvent deuterium isotope effects on kc and kc/Km. pH dependencies of kc/Km were determined for the SLN-catalyzed hydrolysis of three peptides: Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Nle-NH2,Arg-Pro-Ala-Pro-Gln-Gln- Phe-Phe - Gly-Leu-NleNH2, and N-acetyl-Arg-Pro-Ala-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Nle-NH2 (cleavage at Gln-Phe bond). The pH dependencies are all bell-shaped with shoulders that extend from pH 7.5 to 8.5. The existence of a shoulder indicates that the reaction mechanism involves at least two routes to products. These curves are governed by three proton ionizations with pKa values of 5.4, 6.1, and 9.5. The solvent isotope effect measurements provided the following values: D(kc/Km) = 0.80 +/- 0.05 and D(kc) = 1.58 +/- 0.05. That D(kc/Km) and D(kc) are different suggests that the rate-limiting transition states for the processes governed by kc/Km and kc cannot be the same. We use these results, together with analogy to thermolysin catalysis, to develop a mechanism for SLN catalysis. 相似文献
16.
Protein arginine methyltransferases (PRMTs) catalyze the transfer of methyl groups from S-adenosylmethionine (SAM) to the guanidinium group of arginine residues in a number of important cell signaling proteins. PRMT1 is the founding member of this family, and its activity appears to be dysregulated in heart disease and cancer. To begin to characterize the catalytic mechanism of this isozyme, we assessed the effects of mutating a number of highly conserved active site residues (i.e., Y39, R54, E100, E144, E153, M155, and H293), which are believed to play key roles in SAM recognition, substrate binding, and catalysis. The results of these studies, as well as pH-rate studies, and the determination of solvent isotope effects (SIEs) indicate that M155 plays a critical role in both SAM binding and the processivity of the reaction but is not responsible for the regiospecific formation of asymmetrically dimethylated arginine (ADMA). Additionally, mutagenesis studies on H293, combined with pH studies and the lack of a normal SIE, do not support a role for this residue as a general base. Furthermore, the lack of a normal SIE with either the wild type or catalytically impaired mutants suggests that general acid/base catalysis is not important for promoting methyl transfer. This result, combined with the fact that the E144A/E153A double mutant retains considerably more activity then the single mutants alone, suggests that the PRMT1-catalyzed reaction is primarily driven by bringing the substrate guanidinium into the proximity of the S-methyl group of SAM and that the prior deprotonation of the substrate guanidinium is not required for methyl transfer. 相似文献
17.
Onuoha SC Mukund SR Coulstock ET Sengerovà B Shaw J McLaughlin SH Jackson SE 《Journal of molecular biology》2007,372(2):287-297
Heat shock protein 90 (Hsp90) is a molecular chaperone that is required for the maturation and activation of a number of client proteins, many of which are involved in cancer development. The ansamycin family of natural products and their derivatives, such as geldanamycin (GA), are well-known inhibitors of the essential ATPase activity of Hsp90. Despite structural studies on the complexes of ansamycin derivatives with the ATPase domain of Hsp90, certain aspects of their inhibitory mechanism remain unresolved. For example, it is known that GA in solution exists in an extended conformation with a trans amide bond; however, it binds to Hsp90 in a significantly more compact conformation with a cis amide bond. GA and its derivatives have been shown to bind to Hsp90 with low micromolar affinity in vitro, in contrast to the low nanomolar anti-proliferative activity that these drugs exhibit in vivo. In addition, they show selectivity towards tumour cells. We have studied both the equilibrium binding, and the association and dissociation kinetics of GA derivative, 17-DMAG, and the fluorescently labelled analogue BDGA to both wild-type and mutant Hsp90. The mutants were made in order to test the hypothesis that conserved residues near the ATP-binding site may catalyse the trans-cis isomerisation of GA. Our results show that Hsp90 does not catalyse the trans-cis isomerisation of GA, and suggests that there is no isomerisation step before binding to Hsp90. Experiments with BDGA measured over a wide range of conditions, in the absence and in the presence of reducing agents, confirm recent studies that have suggested that the reduced dihydroquinone form of the drug binds to Hsp90 considerably more tightly than the non-reduced quinone species. 相似文献
18.
Muhammad Akhtar Michael R. Calder David L. Corina J. Neville Wright 《The Biochemical journal》1982,201(3):569-580
Mechanistic aspects of the biosynthesis of oestrogen have been studied with a microsomal preparation from full-term human placenta. The overall transformation, termed the aromatization process, involves three steps using O2 and NADPH, in which the C-19 methyl group of an androgen is oxidised to formic acid with concomitant production of the aromatic ring of oestrogen: [Formula: see text] To study the mechanism of this process in terms of the involvement of the oxygen atoms, a number of labelled precursors were synthesized. Notable amongst these were 19-hydroxy-4-androstene-3,17-dione (II) and 19-oxo-4-androstene-3,17-dione (IV) in which the C-19 was labelled with 2H in addition to 18O. In order to follow the fate of the labelled atoms at C-19 of (II) and (IV) during the aromatization, the formic acid released from C-19 was benzylated and analysed by mass spectrometry. Experimental procedures were devised to minimize the exchange of oxygen atoms in substrates and product with oxygens of the medium. In the conversion of the 19-[18O] compounds of types (II) and (IV) into 3-hydroxy-1,3,5-(10)-oestratriene-17-one (V, oestrone), it was found that the formic acid from C-19 retained the original substrate oxygen. When the equivalent 16O substrates were aromatized under 18O2, the formic acid from both substrates contained one atom of 18O. It is argued that in the conversion of the 19-hydroxy compound (II) into the 19-oxo compound (IV), the C-19 oxygen of the former remains intact and that one atom of oxygen from O2 is incorporated into formic acid during the conversion of the 19-oxo compound (IV) into oestrogen. This conclusion was further substantiated by demonstrating that in the aromatization of 4-androstene-3,17-dione (I), both the oxygen atoms in the formic acid originated from molecular oxygen. 10β-Hydroxy-4-oestrene-3,17-dione formate, a possible intermediate in the aromatization, was synthesized and shown not to be converted into oestrogen. In the light of the cumulative evidence available to date, stereochemical aspects of the conversion of the 19-hydroxy compound (II) into the 19-oxo compound (IV), and mechanistic features of the C-10–C-19 bond cleavage step during the conversion of the 19-oxo compound (IV) into oestrogen are discussed. 相似文献
19.
The roles of three conserved active site carboxylic acids (D197, E233, and D300) in the catalytic mechanism of human pancreatic alpha-amylase (HPA) were studied by utilizing site-directed mutagenesis in combination with structural and kinetic analyses of the resultant enzymes. All three residues were mutated to both alanine and the respective amide, and a double alanine mutant (E233A/D300A) was also generated. Structural analyses demonstrated that there were no significant differences in global fold for the mutant enzymes. Kinetic analyses were performed on the mutants, utilizing a range of substrates. All results suggested that D197 was the nucleophile, as virtually all activity (>10(5)-fold decrease in k(cat) values) was lost for the enzymes mutated at this position when assayed with several substrates. The significantly greater second-order rate constant of E233 mutants on "activated" substrates (k(cat)/K(m) value for alpha-maltotriosyl fluoride = 15 s(-)(1) mM(-)(1)) compared with "unactivated" substrates (k(cat)/K(m) value for maltopentaose = 0.0030 s(-)(1) mM(-)(1)) strongly suggested that E233 is the general acid catalyst, as did the pH-activity profiles. Transglycosylation was favored over hydrolysis for the reactions of several of the enzymes mutated at D300. At the least, this suggests an overall impairment of the catalytic mechanism where the reaction then proceeds using the better acceptor (oligosaccharide instead of water). This may also suggest that D300 plays a crucial role in enzymic interactions with the nucleophilic water during the hydrolysis of the glycosidic bond. 相似文献