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1.
Kondo KI Adachi H Shitara E Kojima F Nishimura Y 《Bioorganic & medicinal chemistry》2001,9(5):1091-1095
The relationships between structures and inhibitory activities of glycosidase inhibitors of gem-diamine 1-N-iminosugars in media of enzyme assays have been investigated. It has been proved that gem-diamine 1-N-iminosugar smoothly undergoes a structural change to a hydrated ketone or its derivative via a hemiaminal in the media (pH 5.0-6.3), and that the products generated in the media as well as the parent gem-diamine 1-N-iminosugars potently inhibit glycosidases. 相似文献
2.
A variety of gem-diamine 1-N-iminosugars related to L-iduronic acid were synthesized and evaluated as inhibitors of heparan sulfate uronyl 2-O-sulfotransferase using an in vitro enzyme assay. Two iminosugars containing guanidino groups acted as potent in vitro inhibitors of the enzyme. 相似文献
3.
Phillips RS Demidkina TV Zakomirdina LN Bruno S Ronda L Mozzarelli A 《The Journal of biological chemistry》2002,277(24):21592-21597
The binding of substrates and inhibitors to wild-type Proteus vulgaris tryptophan indole-lyase and to wild type and Y71F Citrobacter freundii tyrosine phenol-lyase was investigated in the crystalline state by polarized absorption microspectrophotometry. Oxindolyl-lalanine binds to tryptophan indole-lyase crystals to accumulate predominantly a stable quinonoid intermediate absorbing at 502 nm with a dissociation constant of 35 microm, approximately 10-fold higher than that in solution. l-Trp or l-Ser react with tryptophan indole-lyase crystals to give, as in solution, a mixture of external aldimine and quinonoid intermediates and gem-diamine and external aldimine intermediates, respectively. Different from previous solution studies (Phillips, R. S., Sundararju, B., & Faleev, N. G. (2000) J. Am. Chem. Soc. 122, 1008-1114), the reaction of benzimidazole and l-Trp or l-Ser with tryptophan indole-lyase crystals does not result in the formation of an alpha-aminoacrylate intermediate, suggesting that the crystal lattice might prevent a ligand-induced conformational change associated with this catalytic step. Wild-type tyrosine phenol-lyase crystals bind l-Met and l-Phe to form mixtures of external aldimine and quinonoid intermediates as in solution. A stable quinonoid intermediate with lambda(max) at 502 nm is accumulated in the reaction of crystals of Y71F tyrosine phenol-lyase, an inactive mutant, with 3-F-l-Tyr with a dissociation constant of 1 mm, approximately 10-fold higher than that in solution. The stability exhibited by the quinonoid intermediates formed both by wild-type tryptophan indole-lyase and by wild type and Y71F tyrosine phenol-lyase crystals demonstrates that they are suitable for structural determination by x-ray crystallography, thus allowing the elucidation of a key species of pyridoxal 5'-phosphate-dependent enzyme catalysis. 相似文献
4.
Sivaraman J Li Y Larocque R Schrag JD Cygler M Matte A 《Journal of molecular biology》2001,311(4):761-776
The biosynthesis of histidine is a central metabolic process in organisms ranging from bacteria to yeast and plants. The seventh step in the synthesis of histidine within eubacteria is carried out by a pyridoxal-5'-phosphate (PLP)-dependent l-histidinol phosphate aminotransferase (HisC, EC 2.6.1.9). Here, we report the crystal structure of l-histidinol phosphate aminotransferase from Escherichia coli, as a complex with pyridoxamine-5'-phosphate (PMP) at 1.5 A resolution, as the internal aldimine with PLP, and in a covalent, tetrahedral complex consisting of PLP and l-histidinol phosphate attached to Lys214, both at 2.2 A resolution. This covalent complex resembles, in structural terms, the gem-diamine intermediate that is formed transiently during conversion of the internal to external aldimine.HisC is a dimeric enzyme with a mass of approximately 80 kDa. Like most PLP-dependent enzymes, each HisC monomer consists of two domains, a larger PLP-binding domain having an alpha/beta/alpha topology, and a smaller domain. An N-terminal arm contributes to the dimerization of the two monomers. The PLP-binding domain of HisC shows weak sequence similarity, but significant structural similarity with the PLP-binding domains of a number of PLP-dependent enzymes. Residues that interact with the PLP cofactor, including Tyr55, Asn157, Asp184, Tyr187, Ser213, Lys214 and Arg222, are conserved in the family of aspartate, tyrosine and histidinol phosphate aminotransferases. The imidazole ring of l-histidinol phosphate is bound, in part, through a hydrogen bond with Tyr110, a residue that is substituted by Phe in the broad substrate specific HisC enzymes from Zymomonas mobilis and Bacillus subtilis.Comparison of the structures of the HisC internal aldimine, the PMP complex and the HisC l-histidinol phosphate complex reveal minimal changes in protein or ligand structure. Proton transfer, required for conversion of the gem-diamine to the external aldimine, does not appear to be limited by the distance between substrate and lysine amino groups. We propose that the tetrahedral complex has resulted from non-productive binding of l-histidinol phosphate soaked into the HisC crystals, resulting in its inability to be converted to the external aldimine at the HisC active site. 相似文献
5.
We have examined the binding of oxindolyl-L-alanine, (3R)-2,3-dihydro-L-tryptophan, L-homophenylalanine, and N1-methyl-L-tryptophan to tryptophan indole-lyase (tryptophanase) from Escherichia coli by using rapid-scanning and single-wavelength stopped-flow kinetic techniques. Rate constants for the reactions were determined by fitting the concentration dependencies of relaxations to either linear (pseudo-first-order) or hyperbolic (rapid second-order followed by slow first-order) equations. The reaction with oxindolyl-L-alanine forms a quinonoid intermediate that exhibits a strong peak at 506 nm. This species is formed more rapidly than with the other analogues (84.5 s-1) and is reprotonated very slowly (0.2 s-1). Reaction with L-homophenylalanine also forms a quinonoid intermediate with a strong peak at 508 nm, but the rate constant for its formation is slower (6.9 s-1). The reaction with L-homophenylalanine exhibits a transient intermediate absorbing at about 340 nm that decays at the same rate as the quinonoid peak forms and that may be a gem-diamine. Tryptophan indole-lyase reacts with (3R)-2,3-dihydro-L-tryptophan much more slowly to form a moderately intense quinonoid peak at 510 nm, and a transient intermediate absorbing at about 350 nm is also formed. The species formed in the reaction of N1-methyl-L-tryptophan exhibits a peak at 425 nm and a very weak quinonoid absorption peak at 506 nm, which is formed at less than 4 s-1.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
6.
Karthikeyan S Zhou Q Zhao Z Kao CL Tao Z Robinson H Liu HW Zhang H 《Biochemistry》2004,43(42):13328-13339
1-Aminocyclopropane-1-carboxylate (ACC) deaminase is a pyridoxal 5'-phosphate (PLP) dependent enzyme catalyzing the opening of the cyclopropane ring of ACC to give alpha-ketobutyric acid and ammonia as the products. This ring cleavage reaction is unusual because the substrate, ACC, contains no abstractable alpha-proton and the carboxyl group is retained in the product. How the reaction is initiated to generate an alpha-carbanionic intermediate, which is the common entry for most PLP-dependent reactions, is not obvious. To gain insight into this unusual ring-opening reaction, we have solved the crystal structures of ACC deaminase from Pseudomonas sp. ACP in complex with substrate ACC, an inhibitor, 1-aminocyclopropane-1-phosphonate (ACP), the product alpha-ketobutyrate, and two d-amino acids. Several notable observations of these structural studies include the following: (1) a typically elusive gem-diamine intermediate is trapped in the enzyme complex with ACC or ACP; (2) Tyr294 is in close proximity (3.0 A) to the pro-S methylene carbon of ACC in the gem-diamine complexes, implicating a direct role of this residue in the ring-opening reaction; (3) Tyr294 may also be responsible for the abstraction of the alpha-proton from d-amino acids, a prelude to the subsequent deamination reaction; (4) the steric hindrance precludes accessibility of active site functional groups to the l-amino acid substrates and may account for the stereospecificity of this enzyme toward d-amino acids. These structural data provide evidence favoring a mechanism in which the ring cleavage is induced by a nucleophilic attack at the pro-S beta-methylene carbon of ACC, with Tyr294 as the nucleophile. However, these observations are also consistent with an alternative mechanistic possibility in which the ring opening is acid-catalyzed and may be facilitated by charge relay through PLP, where Tyr294 functions as a general acid. The results of mutagenesis studies corroborated the assigned critical role for Tyr294 in the catalysis. 相似文献
7.
Jabgunde AM Kalamkar NB Chavan ST Sabharwal SG Dhavale DD 《Bioorganic & medicinal chemistry》2011,19(19):5912-5915
New six- and seven-membered 1-N-iminosugars were prepared from d-glucose by the stereoselective Michael addition of nitromethane to d-glucose derived α,β-unsaturated ester A followed by one pot reduction of nitro/ester functionality and subsequent amine protection to get N-Cbz protected aminol 6. Hydrolysis of 1,2-acetonide and reductive aminocyclization gave seven membered 1-N-iminosugar 5b. While, hydrolysis of 1,2-acetonide followed by NaIO(4) oxidative cleavage and hydrogenation using 10% Pd(OH)(2)/C, H(2) gave six membered 1-N-iminosugar 4a; the hydrogenation using 10% Pd/C-H(2) however, gave N-methyl substituted 1-N-iminosugar 4b. The hydrochloride salts of 4a/4b and 5b were found to be specific α-galactosidase and moderate α-glucosidae inhibitors, respectively, in micro molar range. 相似文献
8.
Ornithine decarboxylase (ODC) is an obligate homodimer that catalyzes the pyridoxal 5'-phosphate-dependent decarboxylation of l-ornithine to putrescine, a vital step in polyamine biosynthesis. A previous mutagenic analysis of the ODC dimer interface identified several residues that were distant from the active site yet had a greater impact on catalytic activity than on dimer stability [Myers, D. P., et al. (2001) Biochemistry 40, 13230-13236]. To better understand the basis of this phenomenon, the structure of the Trypanosoma brucei ODC mutant K294A was determined to 2.15 A resolution in complex with the substrate analogue d-ornithine. This residue is distant from the reactive center (>10 A from the PLP Schiff base), and its mutation reduced catalytic efficiency by 3 kcal/mol. The X-ray structure demonstrates that the mutation increases the disorder of residues Leu-166-Ala-172 (Lys-169 loop), which normally form interactions with Lys-294 across the dimer interface. In turn, the Lys-169 loop forms interactions with the active site, suggesting that the reduced catalytic efficiency is mediated by the decreased stability of this loop. The extent of disorder varies in the four Lys-169 loops in the asymmetric unit, suggesting that the mutation has led to an increase in the population of inactive conformations. The structure also reveals that the mutation has affected the nature of the ligand-bound species. Each of the four active sites contains unusual ligands. The electron density suggests one active site contains a gem-diamine intermediate with d-ornithine; the second has density consistent with a tetrahedral adduct with glycine, and the remaining two contain tetrahedral adducts of PLP, Lys-69, and water (or hydroxide). These data also suggest that the structure is less constrained in the mutant enzyme. The observation of a gem-diamine intermediate provides insight into the conformational changes that occur during the ODC catalytic cycle. 相似文献
9.
In pre-steady-state, fast-quench kinetic analysis, the tertiary-stabilized hammerhead ribozyme "RzB" cleaves its substrate RNA with maximal measured k (obs) values of approximately 3000 min(-1) in 1 mM Mn(2+) and approximately 780 min(-1) in 1 mM Mg(2+) at 37 degrees C (pH 7.4). Apparent pKa for the catalytic general base is approximately 7.8-8.5, independent of the corresponding metal hydrate pKa, suggesting potential involvement of a nucleobase as general base as suggested previously from nucleobase substitution studies. The pH-rate profile is bell-shaped for Cd(2+), for which the general catalytic acid has a pKa of 7.3 +/- 0.1. Simulations of the pH-rate relation suggest a pKa for the general catalytic acid to be approximately 9.5 in Mn(2+) and >9.5 in Mg(2+). The acid pKa's follow the trend in the pKa of the hydrated metal ions but are displaced by approximately 1-2 pH units in the presence of Cd(2+) and Mn(2+). One possible explanation for this trend is direct metal ion coordination with a nucleobase, which then acts as general acid. 相似文献
10.
11.
Rajaram V Bhavani BS Kaul P Prakash V Appaji Rao N Savithri HS Murthy MR 《The FEBS journal》2007,274(16):4148-4160
Serine hydroxymethyltransferase (SHMT) belongs to the alpha-family of pyridoxal 5'-phosphate-dependent enzymes and catalyzes the reversible conversion of L-Ser and tetrahydrofolate to Gly and 5,10-methylene tetrahydrofolate. 5,10-Methylene tetrahydrofolate serves as a source of one-carbon fragment in many biological processes. SHMT also catalyzes the tetrahydrofolate-independent conversion of L-allo-Thr to Gly and acetaldehyde. The crystal structure of Bacillus stearothermophilus SHMT (bsSHMT) suggested that E53 interacts with the substrate, L-Ser and tetrahydrofolate. To elucidate the role of E53, it was mutated to Q and structural and biochemical studies were carried out with the mutant enzyme. The internal aldimine structure of E53QbsSHMT was similar to that of the wild-type enzyme, except for significant changes at Q53, Y60 and Y61. The carboxyl of Gly and side chain of L-Ser were in two conformations in the respective external aldimine structures. The mutant enzyme was completely inactive for tetrahydrofolate-dependent cleavage of L-Ser, whereas there was a 1.5-fold increase in the rate of tetrahydrofolate-independent reaction with L-allo-Thr. The results obtained from these studies suggest that E53 plays an essential role in tetrahydrofolate/5-formyl tetrahydrofolate binding and in the proper positioning of Cbeta of L-Ser for direct attack by N5 of tetrahydrofolate. Most interestingly, the structure of the complex obtained by cocrystallization of E53QbsSHMT with Gly and 5-formyl tetrahydrofolate revealed the gem-diamine form of pyridoxal 5'-phosphate bound to Gly and active site Lys. However, density for 5-formyl tetrahydrofolate was not observed. Gly carboxylate was in a single conformation, whereas pyridoxal 5'-phosphate had two distinct conformations. The differences between the structures of this complex and Gly external aldimine suggest that the changes induced by initial binding of 5-formyl tetrahydrofolate are retained even though 5-formyl tetrahydrofolate is absent in the final structure. Spectral studies carried out with this mutant enzyme also suggest that 5-formyl tetrahydrofolate binds to the E53QbsSHMT-Gly complex forming a quinonoid intermediate and falls off within 4 h of dialysis, leaving behind the mutant enzyme in the gem-diamine form. This is the first report to provide direct evidence for enzyme memory based on the crystal structure of enzyme complexes. 相似文献
12.
The amino-terminal peptides of human prothrombin corresponding to residues 1-51 and 52-156 have been isolated from a thrombin digest of prothrombin fragment 1. The products of digestion were purified by means of barium citrate and ammonium sulfate precipitations, followed by gel filtration and hydroxyapatite chromatographies. They were identified by their molecular sizes as well as their amino acid compositions. Peptides 1-51 (F1A) and 52-156 (F1B) were used as affinity ligands for the isolation of antibody populations from antisera that were elicited against human prothrombin or prothrombin fragment 1. These antibody populations displayed restricted specificity for the respective ligands as shown by competitive radioimmunoassays. They were used to study the conformational changes in prothrombin and fragment 1. The F1A-specific antibody populations detected a conformational change which is stabilized by calcium ions and which has a transition midpoint at approximately 0.2 mM calcium ion concentration. The F1B-specific antibody populations identified a different conformational change which is destabilized by calcium ions and which has a transition midpoint at approximately 0.5 mM calcium. 相似文献
13.
Peter Guengerich F Martin MV McCormick WA Nguyen LP Glover E Bradfield CA 《Archives of biochemistry and biophysics》2004,423(2):309-316
Indigo and indirubin have been reported to be present at low levels in human urine. The possibility that indigoids are physiological ligands of the aryl hydrocarbon receptor (AhR) has been suggested by initial studies in yeast, where indirubin was found to be 50 times more potent than 2,3,7,8-tetrachlorodibenzo[p]dioxin (TCDD), and indigo was found to be equipotent. To demonstrate that these indigoids are bona fide agonists in mammalian systems, we employed a number of in vitro and in vivo measures of AhR agonist potency. In a hepatoma cell reporter system, indigo yielded an EC50 of approximately 5x10(-6)M (indirubin 3' -oxime EC50 approximately 5x10(-7)M, indirubin EC50 approximately 1x10(-7)M). A comparison of these EC50 values with that of 2,3,7,8-tetrachlorodibenzofuran (TCDBF) ( approximately 3x10(-9)M) indicated that these compounds are less potent than classic halogenated-dibenzofurans or -dibenzo-p-dioxins. Competitive binding assays for AhR occupancy showed similar IC50 values for indirubin and TCDBF ( approximately 2x10(-9) and 5x10(-9)M), with the IC50 values of indigo and indirubin 3' -oxime being approximately 10-fold higher. When rats were treated with these indigoids in the range of 1.5-50mg/kg, induction of hepatic cytochrome P450 1A1 was detected. Differences in the rank-order of potency observed in vivo and in vitro could, in part, be explained by metabolism. Although their biological potencies are not as high as has been previously suggested, collectively the results show that these indole-derived pigments are agonists of AhR in vivo. The in vivo results suggest that solubility, distribution, and metabolism influence the response to the compounds. 相似文献
14.
The reactions of 2,3-dihydro-L-tryptophan (DHT) and oxindolyl-L-alanine (OXA) with tryptophan synthase have been investigated by rapid-scanning stopped-flow (RSSF) spectroscopy and by the concentration dependence of rates measured by single-wavelength stopped-flow (SWSF) spectroscopy. The RSSF spectral changes for DHT and OXA show the disappearance of the internal aldimine (lambda max 412 nm), the formation and decay of intermediates absorbing less than or equal to 340 nm, and the appearance of the quinonoid (lambda max 492 and 480 nm, respectively). Rate constants determined by SWSF were either well resolved (i.e., k1[DHT], k-1 greater than k2, k-2 greater than k3, k-3) or indicative of a tightly coupled system (i.e., k1[OXA], k-1 greater than or equal to k2, k-2 greater than k3, k-3). The RSSF spectral changes and SWSF kinetic studies together with computer simulations of the kinetic time courses are consistent with a mechanism that includes formation of a bleached species. Detection of these shorter wavelength species in the reactions of OXA and DHT indicates that substrate analogues with tetrahedral geometry at C-3 induce new protein-substrate interactions that result in the accumulation of species not previously detected in the tryptophan synthase system. The bleached species with lambda max less than or equal to 340 nm are proposed as the gem-diamine intermediates. 相似文献
15.
The concentration dependence of the chemical shifts for the hydrogens H-2, H-8 and H-1' of ITP and for H-8 and H-1' of GTP has been measured in D2O at 25 degrees C under several degrees of protonation in the pD range 1.2-8.4. For reasons of comparison, inosine and guanosine have been included in the study The results are consistent with the isodesmic model of indefinite noncooperative stacking. The association constants for the nucleosides (Ns) inosine and guanosine decrease with increasing protonation: Ns greater than D(Ns)+/Ns in a 1:1 ratio greater than D(Ns)+. In contrast, a maximum is observed with ITP and GTP; the stacking tendency of GTP following the series: GTP4- less than or equal to D(GTP)3- (K approximately 0.7 M-1) less than D(GTP)3-/D2(GTP)2- in a 1:1 ratio (K approximately 2.9 M-1) greater than D2(GTP)2- greater than D3(GTP)- (K approximately 1.5 M-1). The order of the series with ITP corresponds to that with GTP, but the association constants are slightly smaller. At the maximum of the self-association tendency the triphosphate residue has only a minor influence; this follows from the fact that the association constants for the 1:1 ratios of Ino/D(Ino)+ and D(ITP)3-/D2(ITP)2- are identical within experimental error; this holds also for Guo/D(Guo)+ and D(GTP)3-/D2(GTP)2-; in all these pairs the K-7 site is 50% protonated. Comparison of the association constant for the deprotonated species shows that here charge effects, i.e. repulsion between the negatively charged triphosphate chains, are important: Ino (K approximately 3.3 M-1) greater than ITP4- (K approximately 0.4 M-1) and Guo (K approximately 8 M-1) greater than GTP4- (K approximately 0.8 M-1). In addition the series holds: Ado (K approximately 15 M-1) greater than Guo greater than Ino. However, most important is the comparison of the ITP and GTP series with previous data for ATP: ATP4- (K approximately 1.3 M-1) less than D(ATP)3- (2.1 M-1) less than 1:1 ratio of D(ATP)3-/D2(ATP)2- (6 M-1) much less than D2(ATP)2- (approximately 200 M-1) much greater than D3(ATP)- (K less than or equal to 17 M-1).(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
16.
The proliferation parameters of the Walker carcinoma were estimated from both in vivo and in vitro measurements. tthe transplantable Walker carcinoma 256 was grown in male inbred BD1 rats. During exponential growth, 5--6 days after transplantation, a PLM curve was performed, yielding estimates of TC approximately equal to 18-0 hr, TS approximately equal to 6-4 hr, TG2+M approximately equal to 4-1 hr. With the double labelling technique in vitro under 2-2 atm oxygen we obtained: TC approximately equal to 18-2 hr, TS approximately equal to 8-2 hr, TG2+M approximately equal to 2-0 hr. From pulse cytophotometry DNA content histograms the fractions of cells in the cell cycle phases were calculated using a computer program: fG1 approximately equal to (47-6 +/- 1-1)%, fS approximately equal to (34-1 +/- 1-0)%, fG2+M approximately equal to (18-3 +/- 1-5)%. These fractions remained constant between the fifth and the twelfth day after transplantation. At that time the tumour growth had already slowed down appreciably. The growth fraction determined by repetitive labelling was 0.96 on the fifth and 0-93 on the seventh and eleventh day. The cell loss factor was phi approximately equal to 17% during exponential tumor growth and increased to about 100% between the tenth and twelfth day. The agreement of the cell kinetic data determined by autoradiography from solid tumours in vivo (PLM, continuous labelling) and autoradiography as well as pulse cytophotometry from in vitro experiments (excised material) was satisfactory. 相似文献
17.
Diffusion of water in the endosperm tissue of wheat grains as studied by pulsed field gradient nuclear magnetic resonance.
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Pulsed field gradient nuclear magnetic resonance has been used to measure water self-diffusion coefficients in the endosperm tissue of wheat grains as a function of the tissue water content. A model that confines the water molecules to a randomly oriented array of capillaries with both transverse dimension less than 100 nm has been used to fit the data and give a unique diffusion coefficient at each water content. The diffusion rates vary from 1.8 x 10(-10) m2s-1 at the lowest to 1.2 x 10(-9) m2s-1 at the highest moisture content. This variation can be explained in terms of an increase in water film thickness from approximately 0.5 to approximately 2.5 nm over the moisture range investigated (200-360 mg g-1). 相似文献
18.
Asn185 is an invariant residue in all known sequences of TPL and of closely related tryptophanase and it may be aligned with the Asn194 in aspartate aminotransferase. According to X-ray data, in the holoenzyme and in the Michaelis complex Asn185 does not interact with the cofactor pyridoxal 5'-phosphate, but in the external aldimine a conformational change occurs which is accompanied by formation of a hydrogen bond between Asn185 and the oxygen atom in position 3 of the cofactor. The substitution of Asn185 in TPL by alanine results in a mutant N185A TPL of moderate residual activity (2%) with respect to adequate substrates, L-tyrosine and 3-fluoro-L-tyrosine. The affinities of the mutant enzyme for various amino acid substrates and inhibitors, studied by both steady-state and rapid kinetic techniques, were lower than for the wild-type TPL. This effect mainly results from destabilization of the quinonoid intermediate, and it is therefore concluded that the hydrogen bond between Asn185 and the oxygen at the C-3 position of the cofactor is maintained in the quinonoid intermediate. The relative destabilization of the quinonoid intermediate and external aldimine leads to the formation of large amounts of gem-diamine in reactions of N185A TPL with 3-fluoro-L-tyrosine and L-phenylalanine. For the reaction with 3-fluoro-L-tyrosine it was first possible to determine kinetic parameters of gem-diamine formation by the stopped-flow method. For the reactions of N185A TPL with substrates bearing good leaving groups the observed values of k(cat) could be accounted for by taking into consideration two effects: the decrease in the quinonoid content under steady-state conditions and the increase in the quinonoid reactivity in a beta-elimination reaction. Both effects are due to destabilization of the quinonoid and they counterbalance each other. Multiple kinetic isotope effect studies on the reactions of N185A TPL with suitable substrates, L-tyrosine and 3-fluoro-L-tyrosine, show that the principal mechanism of catalysis, suggested previously for the wild-type enzyme, does not change. In the framework of this mechanism the observed considerable decrease in k(cat) values for reactions of N185A TPL with L-tyrosine and 3-fluoro-L-tyrosine may be ascribed to participation of Asn185 in additional stabilization of the keto quinonoid intermediate. 相似文献
19.
Our studies of the reaction mechanism of cystathionine beta-synthase from Saccharomyces cerevisiae (yeast) are facilitated by the spectroscopic properties of the pyridoxal phosphate coenzyme that forms a series of intermediates in the reaction of L-serine and L-homocysteine to form L-cystathionine. To characterize these reaction intermediates, we have carried out rapid-scanning stopped-flow and single-wavelength stopped-flow kinetic measurements under pre-steady-state conditions, as well as circular dichroism and fluorescence spectroscopy under steady-state conditions. We find that the gem-diamine and external aldimine of aminoacrylate are the primary intermediates in the forward half-reaction with L-serine and that the external aldimine of aminoacrylate or its complex with L-homocysteine is the primary intermediate in the reverse half-reaction with L-cystathionine. The second forward half-reaction of aminoacrylate with L-homocysteine is rapid. No primary kinetic isotope effect was obtained in the forward half-reaction with L-serine. The results provide evidence (1) that the formation of the external aldimine of L-serine is faster than the formation of the aminoacrylate intermediate, (2) that aminoacrylate is formed by the concerted removal of the alpha-proton and the hydroxyl group of L-serine, and (3) that the rate of the overall reaction is rate-limited by the conversion of aminoacrylate to L-cystathionine. We compare our results with cystathionine beta-synthase with those of related investigations of tryptophan synthase and O-acetylserine sulfhydrylase. 相似文献
20.
Solution structure and dynamics of the plasminogen kringle 2-AMCHA complex: 3(1)-helix in homologous domains 总被引:1,自引:0,他引:1
The kringle 2 (K2) module of human plasminogen (Pgn) binds L-lysine and analogous zwitterionic compounds, such as the antifibronolytic agent trans-(aminomethyl)cyclohexanecarboxylic acid (AMCHA). Far-UV CD and NMR spectra reveal little conformational change in K2 upon ligand binding. However, retarded (1)H-(2)H isotope exchange kinetics induced by AMCHA indicate stabilization of the K2 conformation by the ligand. Assessment of secondary structure content from CD spectra yields approximately 26% beta-STRAND, approximately 13% beta-TURN, approximately 15% 3(1)-HELIX, and approximately 6% 3(10)-HELIX. The NMR solution conformation of the K2 domain complexed to AMCHA has been determined [heavy atom rmsd = 0.49 +/- 0.09A (BACKBONE) AND 1.02+/- 0.08 (ALL)]. The K2 molecule has overall dimensions of approximately 34.5A times approximately 33.4A times approximately 22.7A . Analogous with the polypeptide outline of homologous domains, K2 contains three short antiparallel beta-sheets (paired strands 15-16/20-21, 24-25/48-49, and 62-64/72-74) and four defined beta-turns (residues 6-9, 16-19, 53-56, AND 67-70). Consistent with the CD analysis, albeit novel in the context of kringle folding, the NMR structure reveals an unpaired beta-strand structured by residues 30-32, a turn of 3(10)-helix compromising residues 38-41, and a 3(1)-helix for residues 21-24 and 74-79. We also identify alignable 3(1)-helices in previously reported homologous kringle structures. Rather high order parameter S(2) values (= approximately 0.85 +/- 0.04) characterize the K2 backbone dynamics. The lowest flexibility is observed for the two inner loop segments of residues 51-63 AND 63-75 (= approximately 0.86-0.87 +/- 0.03). Overhauser connectivities reveal close hydrophobic contacts of the ligand ring with side chains of Tyr(36), Trp(62), Phe(64), Trp(72), AND Leu(74). In most K2 structures, the N atom of AMCHA places itself approximately 3.9 and 4.4A from the anionic groups of Glu(57) and Asp(55), respectively, while its carboxylate group, H-bonded to the Tyr(36) side chain OH(eta), ion-pairs the Arg(71) guanidinium group. Consistent with the preference of K2 for binding 5-aminopentanoic acid over 6-aminohexanoic acid, the positions of the ionic centers within the K2 binding site approach each other approximately 1A closer relative to what is observed in lysine binding sites of homologous Pgn modules. 相似文献