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1.
The kinetics of binding of bovine trypsin to a proteinaceous inhibitor of trypsin from buckwheat seeds (BWI-1a) has been studied. The association rate constant (k(ass)) was 2.2 x 10(6) M-1 x sec-1 and the dissociation rate constant (k(off)) of the enzyme--inhibitor complex was 3.5 x 10(-3) sec-1; the inhibition constant Ki was 1.5 nM. The inhibitor BWI-1a is of the slow, tightly binding type. The mechanism of the inhibition of bovine trypsin by the trypsin inhibitor BWI-1a was studied. The mechanism of inhibition was found to involve two steps according to the kinetic data.  相似文献   

2.
In neutral solution, 5,6-dihydrocytidine undergoes spontaneous deamination (k25 approximately 3.2 x 10(-5) s(-1)) much more rapidly than does cytidine (k25 approximately 3.0 x 10(-10) s(-1)), with a more favorable enthalpy of activation (DeltaDeltaH# = -8.7 kcal/mol) compensated by a less favorable entropy of activation (TDeltaDeltaS# = -1.8 kcal/mol at 25 degrees C). E. coli cytidine deaminase enhances the rate of deamination of 5,6-dihydrocytidine (kcat/k(non) = 4.4 x 10(5)) by enhancing the entropy of activation (DeltaDeltaH# = 0 kcal/mol; TDeltaDeltaS# = +7.6 kcal/mol, at 25 degrees C). Binding of the competitive inhibitor 3,4,5,6-tetrahydrouridine (THU), a stable analogue of 5,6-dihydrocytidine in the transition state for its deamination, is accompanied by a release of enthalpy (DeltaH = -7.1 kcal/mol, TDeltaDeltaS = +2.2 kcal/mol) that approaches the estimated enthalpy of binding of the actual substrate in the transition state for deamination of 5,6-dihydrocytidine (DeltaH = -8.1 kcal/mol, TDeltaDeltaS = +6.0 kcal/mol). Thus, the shortcomings of THU in capturing all of the binding affinity expected of an ideal transition-state analogue reflect a less favorable entropy of association. That difference may arise from the analogue's inability to displace a water molecule from the "leaving group site" at which ammonia is generated in the normal reaction. The effect on binding of removing the 4-OH group from the transition-state analogue THU, to form 3,4,5,6-tetrahydrozebularine (THZ) (DeltaDeltaH = -2.1 kcal/mol, TDeltaDeltaS = -4.4 kcal/mol), is mainly entropic, consistent with the inability of THZ to displace water from the "attacking group site". These results are consistent with earlier indications [Snider, M. J., and Wolfenden, R. (2001) Biochemistry 40, 11364] that site-bound water plays a prominent role in substrate activation and inhibitor binding by cytidine deaminase.  相似文献   

3.
The methionyl-tRNA synthetase from Bacillus stearothermophilus is shown to be a dimer of 2 x 82,000 with identical subunits. It exhibits negative cooperativity in substrate binding and "virtual" halt-of-the-sites reactivity. The enzyme binds only 1 mol of methionine in the absence of other ligands, but several methods show that 2 mol of methionyl adenylate are bound per enzyme dimer. However, one of these adenylates is formed 480 times faster than the other (k1 = 29 sec-1 and k2 = 0.06 sec-1). While the rapid phase of the reaction follows normal saturation kinetics with respect to substrate concentration, the rate of the slow phase is independent of substrate concentrations down to 1 muM. It is suggested that the very slow rate of formation of the second adenylate reflects a rate limiting conformational change which precedes a more rapid chemical step on the second subunit.  相似文献   

4.
The nature of the interaction between Escherichia coli cytidine deaminase and the phosphapyrimidine nucleoside 1 has been studied kinetically and spectrophotometrically. Compound 1 was designed as a transition-state analog, and is a potent, slow-binding inhibitor of cytidine deaminase (Ashley, G. W., and Bartlett, P. A. (1982) Biochem. Biophys. Res. Commun. 108, 1467-1474). We present evidence that the binding of 1 is reversible, with no covalent linkage between the enzyme and 1. At pH 6, the rate of recovery of enzyme activity from dissociation of the E X I complex is strongly dependent on the concentration of E X I, indicating that the inhibitor dissociates reversibly. UV difference spectroscopy reveals that the chromophore of 1 is unaltered on binding to the enzyme, thus eliminating the possibility of reversible, covalent modification of the enzyme. For the binding of the active beta-anomers of 1 to cytidine deaminase, the following kinetic parameters were determined at pH 6: kon = 8300 M-1 S-1, koff = 7.8 X 10(-6) S-1, Ki = 0.9 nM. We were also able to observe and characterize time-dependent inhibition of E. coli cytidine deaminase by tetrahydrouridine, 3. This interaction involves involves initial formation of a loose complex (KD = 1.2 microM), followed by isomerization in a slow step to give a more tightly bound complex (Ki = 0.24 microM) with forward and reverse rate constants kf = 3.81 min-1 and kr = 0.95 min-1, respectively.  相似文献   

5.
Cytidine deamination of nucleic acids underlies diversification of Ig genes and inhibition of retroviral infection, and thus, it would appear to be vital to host defense. The host defense properties of cytidine deamination require two distinct but homologous cytidine deaminases-activation-induced cytidine deaminase and apolipoprotein B-editing cytidine deaminase, subunit 3G. Although cytidine deamination has clear benefits, it might well have biological costs. Uncontrolled cytidine deamination might generate misfolded polypeptides, dominant-negative proteins, or mutations in tumor suppressor genes, and thus contribute to tumor formation. How cytidine deaminases target a given nucleic acid substrate at specific sequences is not understood, and what protects cells from uncontrolled mutagenesis is not known. In this paper, I shall review the functions and regulation of activation-induced cytidine deaminase and apolipoprotein B-editing cytidine deaminase, subunit 3G, and speculate about the basis for site specificity vis-à-vis generalized mutagenesis.  相似文献   

6.
To obtain a clearer understanding of the forces involved in transition state stabilization by Escherichia coli cytidine deaminase, we investigated the thermodynamic changes that accompany substrate binding in the ground state and transition state for substrate hydrolysis. Viscosity studies indicate that the action of cytidine deaminase is not diffusion-limited. Thus, K(m) appears to be a true dissociation constant, and k(cat) describes the chemical reaction of the ES complex, not product release. Enzyme-substrate association is accompanied by a loss of entropy and a somewhat greater release of enthalpy. As the ES complex proceeds to the transition state (ES), there is little further change in entropy, but heat is taken up that almost matches the heat that was released with ES formation. As a result, k(cat)/K(m) (describing the overall conversion of the free substrate to ES is almost invariant with changing temperature. The free energy barrier for the enzyme-catalyzed reaction (k(cat)/K(m)) is much lower than that for the spontaneous reaction (k(non)) (DeltaDeltaG = -21.8 kcal/mol at 25 degrees C). This difference, which also describes the virtual binding affinity of the enzyme for the activated substrate in the transition state (S), is almost entirely enthalpic in origin (DeltaDeltaH = -20.2 kcal/mol), compatible with the formation of hydrogen bonds that stabilize the ES complex. Thus, the transition state affinity of cytidine deaminase increases rapidly with decreasing temperature. When a hydrogen bond between Glu-91 and the 3'-hydroxyl moiety of cytidine is disrupted by truncation of either group, k(cat)/K(m) and transition state affinity are each reduced by a factor of 10(4). This effect of mutation is entirely enthalpic in origin (DeltaDeltaH approximately 7.9 kcal/mol), somewhat offset by a favorable change in the entropy of transition state binding. This increase in entropy is attributed to a loss of constraints on the relative motions of the activated substrate within the ES complex. In an Appendix, some objections to the conventional scheme for transition state binding are discussed.  相似文献   

7.
Cell free extract of nitrate-grown Aspergillus niger NRRL3 catalyzed the hydrolytic deamination of cytidine out of the tested bases, their nucleosides and nucleotides to uridine maximally at pH 7 and at 50 degrees C. The deaminating activity seems to be specific for cytidine, as the extracts could not deaminate AMP, GMP, CMP, adenosine, guanosine, adenine, guanine, and cytosine. Maximum activity of cytidine deaminase was achieved in Tris-HCl buffer at concentration of 0.15 M. Incubation of the extracts at 70 degrees C for 30 minutes in absence of cytidine caused about 70% loss in its activity, while dialysis, freezing and thawing has no effect on the activity. Results indicated the absence of the involvement of SH group(s) in the catalytic site of cytidine deaminase. Uridine competitively inhibited the enzyme activity, while ammonia had no effect. The apparent K(m) value of this enzyme for cytidine was 2.6 x 10(-3) and the Ki value for uridine was 10.06 x 10(-3).  相似文献   

8.
(R)- and (S)-2'-deoxycoformycin, (R)-coformycin, and the corresponding 5'-monophosphates were compared as inhibitors of yeast AMP deaminase. The overall inhibition constants ranged from 4.2 mM for (S)-2'-deoxycoformycin to 10 pM for (R)-coformycin 5'-monophosphate, a difference of 3.8 x 10(8) in affinities. (R)-Coformycin, (R)-2'-deoxycoformycin 5'-monophosphate, and (R)-coformycin 5'-monophosphate exhibited both rapid and slow-onset inhibition. The S inhibitors and (R)-2'-deoxycoformycin exhibited classical competitive inhibition but no time-dependent onset of inhibition. The results indicate that the presence of the 2'-hydroxyl and 5'-phosphate and the R stereochemistry at the C-8 position of the diazepine ring are necessary for the optimum interaction of inhibitors with yeast AMP deaminase. This differs from the results for rabbit muscle AMP deaminase [Frieden C., Kurz, L. C., & Gilbert, H. R. (1980) Biochemistry 19, 5303-5309] and calf intestinal adenosine deaminase [Schramm, V. L., & Baker, D. C. (1985) Biochemistry 24, 641-646], in which a tetrahedral hydroxyl at C-8 in the R stereochemistry is sufficient for slow-onset inhibition with the coformycins. The results suggest that the transition state contains a tetrahedral carbon with the R configuration as a result of the direct attack of an oxygen nucleophile at C-6 of AMP. Slow-onset inhibition of yeast AMP deaminase is consistent with the mechanism [formula: see text] in which the combination of E and I is rapidly reversible. For these inhibitors, Ki varied by a factor of 3 x 10(3), and the overall inhibition constant (Ki*) varied by a factor of 2 x 10(5).(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

9.
Inhibition of beta-site amyloid precursor protein-cleaving enzyme by a statine-based inhibitor has been studied using steady state and stopped-flow methods. A slow onset rate of inhibition has been observed under steady state conditions, and a K(i) of 22 nm has been derived using progress curves analysis. Simulation of stopped-flow protein fluorescence transients provided an estimate of the K(d) for initial inhibitor binding of 660 nm. A two-step inhibition mechanism is proposed, wherein slower "tightening up" of the initial encounter complex occurs. Two hypotheses have been proposed in the literature to address the nature of the slow step in the inhibition of aspartic proteases by peptidomimetic inhibitors: a conformational change related to the "flap" movement and displacement of a catalytic water. We compared substrate and inhibitor binding rates under pre-steady-state conditions. Both ligands are likely to cause flap movement, whereas no catalytic water replacement occurs during substrate binding. Our results suggest that both ligands bind to the enzyme at a rate significantly lower than the diffusion limit, but there are additional rate limitations involved in inhibitor binding, resulting in a k(on) of 3.5 x 10(4) m(-)1 s(-)1 for the inhibitor compared with 3.5 x 10(5) m(-)1 s(-)1 for the substrate. Even though specific intermediate formation steps might be different in the productive inhibitor and substrate binding to beta-site amyloid precursor protein-cleaving enzyme, a similar final optimized conformation is achieved in both cases, as judged by the comparable free energy changes (DeltaDeltaG of 2.01 versus 1.97 kcal/mol) going from the initial to the final enzyme-inhibitor or enzyme-substrate complexes.  相似文献   

10.
We have found that chronically HIV-1(IIIB)-infected H9 cells showed 21-fold resistance to 1-beta-D-arabinofuranosylcytosine (ARA-C) compared with uninfected H9 cells. In the infected H9 cells, a 37% increase of dCTP pool and a 34% increase of dATP were observed, and no alteration of dTTP and dGTP was observed, compared with the uninfected H9 cells. A marked decrease of ARA-CTP generation was observed in the infected H9 cells after 3-h incubation with 0.1-10 microM ARA-C. The level of deoxycytidine kinase activity with ARA-C as substrate was similar in both the infected and the uninfected cells; however, a 37-fold increase of cytidine deaminase activity was observed in the infected H9 cells. These results indicate that the induction of cytidine deaminase activity by HIV-1(IIIB) infection conferred ARA-C resistance to H9 cells. This conclusion was supported by the observation that a marked reversal of ARA-C resistance in the infected H9 cells occurred after treatment with the inhibitor of cytidine deaminase, 3,4,5,6-tetrahydrouridine. The understanding of these cellular alterations in drug sensitivity may facilitate the development of effective therapeutic strategies against HIV-1-infected cells.  相似文献   

11.
Yeast co-expressing rat APOBEC-1 and a fragment of human apolipoprotein B (apoB) mRNA assembled functional editosomes and deaminated C6666 to U in a mooring sequence-dependent fashion. The occurrence of APOBEC-1-complementing proteins suggested a naturally occurring mRNA editing mechanism in yeast. Previously, a hidden Markov model identified seven yeast genes encoding proteins possessing putative zinc-dependent deaminase motifs. Here, only CDD1, a cytidine deaminase, is shown to have the capacity to carry out C→U editing on a reporter mRNA. This is only the second report of a cytidine deaminase that can use mRNA as a substrate. CDD1-dependent editing was growth phase regulated and demonstrated mooring sequence-dependent editing activity. Candidate yeast mRNA substrates were identified based on their homology with the mooring sequence-containing tripartite motif at the editing site of apoB mRNA and their ability to be edited by ectopically expressed APOBEC-1. Naturally occurring yeast mRNAs edited to a significant extent by CDD1 were, however, not detected. We propose that CDD1 be designated an orphan C→U editase until its native RNA substrate, if any, can be identified and that it be added to the CDAR (cytidine deaminase acting on RNA) family of editing enzymes.  相似文献   

12.
The mechanism of slow binding inhibition of 3-hydroxy-3-methylglutaryl- coenzyme A reductase by lovastatin, fluindostatin, and related compounds was studied. Several of these compounds, including lovastatin, were found to be slow binding, while other less potent inhibitors were not. From a comparison of kinetic parameters obtained by steady-state measurements and progress curve analysis, it was concluded that the slow binding inhibitors bind by a mechanism which is more accurately described by biphasic binding than by single-step binding. The overall association rates of the slow binding inhibitors range from 1 x 10(6) to 4 x 10(-7) M-1 s-1, and the dissociation rates are in the range of 10(-3) s-1. The structures of slow binding and reversible inhibitors were compared by using molecular modeling methods. From these comparisons, it was proposed that the slow binding and very potent inhibition of, for instance, lovastatin, is not simply a result of binding of a transition state or reaction intermediate analogue. The various lipophilic groups of the inhibitors that do not seem to be related to structural features of the substrate may also play a crucial role in determining the mechanism of binding of HMGR inhibitors.  相似文献   

13.
The cytidine (C) to uridine (U) editing of apolipoprotein (apo) B mRNA is mediated by tissue-specific, RNA-binding cytidine deaminase APOBEC1. APOBEC1 is structurally homologous to Escherichia coli cytidine deaminase (ECCDA), but has evolved specific features required for RNA substrate binding and editing. A signature sequence for APOBEC1 has been used to identify other members of this family. One of these genes, designated APOBEC2, is found on chromosome 6. Another gene corresponds to the activation-induced deaminase (AID) gene, which is located adjacent to APOBEC1 on chromosome 12. Seven additional genes, or pseudogenes (designated APOBEC3A to 3G), are arrayed in tandem on chromosome 22. Not present in rodents, this locus is apparently an anthropoid-specific expansion of the APOBEC family. The conclusion that these new genes encode orphan C to U RNA-editing enzymes of the APOBEC family comes from similarity in amino acid sequence with APOBEC1, conserved intron/exon organization, tissue-specific expression, homodimerization, and zinc and RNA binding similar to APOBEC1. Tissue-specific expression of these genes in a variety of cell lines, along with other evidence, suggests a role for these enzymes in growth or cell cycle control.  相似文献   

14.
Snider MJ  Wolfenden R 《Biochemistry》2001,40(38):11364-11371
Kinetic measurements have shown that substantial enthalpy changes accompany substrate binding by cytidine deaminase, increasing markedly as the reaction proceeds from the ground state (1/K(m), DeltaH = -13 kcal/mol) to the transition state (1/K(tx), DeltaH = -20 kcal/mol) [Snider, M. J., et al. (2000) Biochemistry 39, 9746-9753]. In the present work, we determined the thermodynamic changes associated with the equilibrium binding of inhibitors by cytidine deaminase by isothermal titration calorimetry and van't Hoff analysis of the temperature dependence of their inhibition constants. The results indicate that the binding of the transition state analogue 3,4-dihydrouridine DeltaH = -21 kcal/mol), like that of the transition state itself (DeltaH = -20 kcal/mol), is associated with a large favorable change in enthalpy. The significantly smaller enthalpy change that accompanies the binding of 3,4-dihydrozebularine (DeltaH = -10 kcal/mol), an analogue of 3,4-dihydrouridine in which a hydrogen atom replaces this inhibitor's 4-OH group, is consistent with the view that polar interactions with the substrate at the site of its chemical transformation play a critical role in reducing the enthalpy of activation for substrate hydrolysis. The entropic shortcomings of 3,4-dihydrouridine, in capturing all of the free energy involved in binding the actual transition state, may arise from its inability to displace a water molecule that occupies the binding site normally occupied by product ammonia.  相似文献   

15.
We have solved the NMR structure of the 31-nucleotide (nt) apoB mRNA stem-loop, a substrate of the cytidine deaminase APOBEC1. We found that the edited base located at the 5' end of the octa-loop is stacked between two adenosines in both the unedited (cytidine 6666) and the edited (uridine 6666) forms and that the rest of the loop is unstructured. The 11-nt "mooring" sequence essential for editing is partially flexible although it is mostly in the stem of the RNA. The octa-loop and the internal loop in the middle of the stem confer this flexibility. These findings shed light on why APOBEC1 alone cannot edit efficiently the cytidine 6666 under physiological conditions, the editing base being buried in the loop and not directly accessible. We also show that APOBEC1 does not specifically bind apoB mRNA and requires the auxiliary factor, APOBEC1 complementary factor (ACF), to edit specifically cytidine 6666. The binding of ACF to both the mooring sequence and APOBEC1 explains the specificity of the reaction. Our NMR study lead us to propose a mechanism in which ACF recognizes first the flexible nucleotides of the mooring sequence (the internal loop and the 3' end octa-loop) and subsequently melts the stem-loop, exposing the amino group of the cytidine 6666 to APOBEC1. Thus, the flexibility of the mooring sequence plays a central role in the RNA recognition by ACF.  相似文献   

16.
A convenient and efficient procedure for the purification of cytidine deaminase (EC 3.5.4.5) from Escherichia coli is reported. The key step involves adsorption of the enzyme from a crude ammonium sulfate fraction onto a cytidine-containing affinity resin, followed by elution with 0.5 M borate buffer. Subsequent chromatography on DEAE-Sepharose results in an overall 1690-fold purification, yielding enzyme with a specific activity of 118 units/mg. Cytidine deaminase has an apparent molecular weight of 54,000 as determined by gel filtration, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows a band at molecular weight 35,000. Cytidine deaminase is inhibited by 5-(chloromercuri)cytidine with kinetic behavior typical of active-site-directed inactivation, with KD = 0.09 mM and kinact = 1.25 min-1. The enzyme is protected against inactivation in the presence of substrate, and the inhibition is reversed with high concentrations of mercaptoethanol. This suggests that inactivation is the result of a mercaptide formation between the mercury and an active-site thiol.  相似文献   

17.
We have analysed a kinetic model of axonal transport by simulating experimental tracer profiles. The existence of three phases of axoplasmic transport is assumed: fast anterograde, slow anterograde and retrograde. Each phase has its characteristic velocity. Transported materials are postulated to shift between these phases. Also catabolism and sequestration of material is allowed for in our model. Thus, we have set up equations which contain axonal transport, diffusion and cross-over terms. The rate constants of material shifts were determined by computer fitting to experimental data. Best-fitted values of the rate constants for transfer of material between the fast and slow phases were both 2 X 10(-5) sec-1, while the rate constants for transfer between the fast and retrograde phases were both 1 X 10(-5) sec-1. The rate constant of material loss from the slow phase to the extracellular space was 1 X 10(-6) sec-1. The material shift between the slow and retrograde phases was negligibly small. These data show that there is exchange of material between the fast and slow phases and between the fast and retrograde phases. However, there is no significant exchange between the slow and retrograde phases. Diffusion was found to have only a minor effect on the profiles. The velocity of the fast anterograde track in cold-blooded animals was predicted to be around 200 mm/day, or, in other words, to be close to experimentally observed values of the fast anterograde component of axonal transport.  相似文献   

18.
Guanine deaminase, a key enzyme in the nucleotide metabolism, catalyzes the hydrolytic deamination of guanine into xanthine. The crystal structure of the 156-residue guanine deaminase from Bacillus subtilis has been solved at 1.17-A resolution. Unexpectedly, the C-terminal segment is swapped to form an intersubunit active site and an intertwined dimer with an extensive interface of 3900 A(2) per monomer. The essential zinc ion is ligated by a water molecule together with His(53), Cys(83), and Cys(86). A transition state analog was modeled into the active site cavity based on the tightly bound imidazole and water molecules, allowing identification of the conserved deamination mechanism and specific substrate recognition by Asp(114) and Tyr(156'). The closed conformation also reveals that substrate binding seals the active site entrance, which is controlled by the C-terminal tail. Therefore, the domain swapping has not only facilitated the dimerization but has also ensured specific substrate recognition. Finally, a detailed structural comparison of the cytidine deaminase superfamily illustrates the functional versatility of the divergent active sites found in the guanine, cytosine, and cytidine deaminases and suggests putative specific substrate-interacting residues for other members such as dCMP deaminases.  相似文献   

19.
15N isotope effects and solvent deuterium isotope effects have been measured for the hydrolytic deamination of cytidine catalyzed by Escherichia coli cytidine deaminase and for the uncatalyzed reaction proceeding spontaneously in neutral solution at elevated temperatures. The primary (15)(V/K) arising from the exocyclic amino group for wild-type cytidine deaminase acting on its natural substrate, cytidine, is 1.0109 (in H(2)O, pH 7.3), 1.0123 (in H(2)O, pH 4.2), and 1.0086 (in D(2)O, pD 7.3). Increasing solvent D(2)O content has no substantial effect on k(cat) but enhances k(cat)/K(m), with a proton inventory showing that the fractionation factors of at least two protons increase markedly during the reaction. Mutant cytidine deaminases with reduced catalytic activity show more pronounced (15)N isotope effects of 1.0124 (Glu91Ala), 1.0134 (His102Ala), and 1.0158 (His102Asn) at pH 7.3 in H(2)O, as expected for processes in which the chemical transformation of the substrate becomes more rate determining. The isotope effect of mutant His102Asn is 1.033 after correcting for protonation of the -NH(2) group, and represents the intrinsic isotope effect on C-N bond cleavage. This result allows an estimation of the forward commitment of the reaction with the wild-type enzyme. The observed (15)N kinetic isotope effect of the pyrimidine N-3, for wild-type cytidine deaminase acting on cytidine, is 0.9879, which is consistent with protonation and rehybidization of N-3 with hydroxide ion attack on the adjacent carbon to create a tetrahedral intermediate. These results show that enzymatic deamination of cytidine proceeds stepwise through a tetrahedral intermediate with ammonia elimination as the major rate-determining step. The primary (15)N isotope effects observed for the uncatalyzed reaction at pH 7 (1.0021) and pH 12.5 (1.0034) were found to be insensitive to changing temperatures between 100 and 185 degrees C. These results show that the uncatalyzed and the enzymatic deaminations of cytidine proceed by similar mechanisms, although the commitment to C-N bond breaking is greater for the spontaneous reaction.  相似文献   

20.
The set of blasticidin S (BS) and blasticidin S deaminase (BSD) is a widely used selectable marker for gene transfer experiments. BSD is a member of the cytidine deaminase (CDA) family; it is a zinc-dependent enzyme with three cysteines and one water molecule as zinc ligands. The crystal structures of BSD were determined in six states (i.e. native, substrate-bound, product-bound, cacodylate-bound, substrate-bound E56Q mutant, and R90K mutant). In the structures, the zinc position and coordination structures vary. The substrate-bound structure shows a large positional and geometrical shift of zinc with a double-headed electron density of the substrate that seems to be assigned to the amino and hydroxyl groups of the substrate and product, respectively. In this intermediate-like structure, the steric hindrance of the hydroxyl group pushes the zinc into the triangular plane consisting of three cysteines with a positional shift of approximately 0.6 A, and the fifth ligand water approaches the opposite direction of the substrate with a shift of 0.4 A. Accordingly, the zinc coordination is changed from tetrahedral to trigonal bipyramidal, and its coordination distance is extended between zinc and its intermediate. The shift of zinc and the recruited water is also observed in the structure of the inactivated E56Q mutant. This novel observation is different in two-cysteine cytidine deaminase Escherichia coli CDA and might be essential for the reaction mechanism in BSD, since it is useful for the easy release of the product by charge compensation and for the structural change of the substrate.  相似文献   

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