Adenosine deaminase prefers a distinct sugar ring conformation for binding and catalysis: kinetic and structural studies

Several recent X-ray crystal structures of adenosine deaminase (ADA) in complex with various adenosine surrogates have illustrated the preferred mode of substrate binding for this enzyme. To define more specific structural details of substrate preferences for binding and catalysis, we have studied the ADA binding efficiencies and deamination kinetics of several synthetic adenosine analogues in which the furanosyl ring is biased toward a particular conformation. NMR solution studies and pseudorotational analyses were used to ascertain the preferred furanose ring puckers (P, nu(MAX)) and rotamer distributions (chi and gamma) of the nucleoside analogues. It was shown that derivatives which are biased toward a "Northern" (3'-endo, N) sugar ring pucker were deaminated up to 65-fold faster and bound more tightly to the enzyme than those that preferred a "Southern" (2'-endo, S) conformation. This behavior, however, could be modulated by other structural factors. Similarly, purine riboside inhibitors of ADA that prefer the N hemisphere were more potent inhibitors than S analogues. These binding propensities were corroborated by detailed molecular modeling studies. Docking of both N- and S-type analogues into the ADA crystal structure coordinates showed that N-type substrates formed a stable complex with ADA, whereas for S-type substrates, it was necessary for the sugar pucker to adjust to a 3'-endo (N-type) conformation to remain in the ADA substrate binding site. These data outline the intricate structural details for optimum binding in the catalytic cleft of ADA.     

Microsomal delta 8,14-sterol delta 14-reductase in higher plants. Characterization and inhibition by analogues of a presumptive carbocationic intermediate of the reduction reaction

An enzymatic assay for delta 8,14-sterol delta 14-reductase, an enzyme involved in sterol biosynthesis, has been developed for the first time in higher plants. The properties of the microsomal enzyme have been established with respect to cofactor requirements, kinetics and substrate specificity. This enzymatic double-bound reduction is thought to proceed through an electrophilic addition mechanism, involving a C14 putative carbonium ion high-energy intermediate. Using this in vitro assay, ammonium and iminium analogues of this cationic intermediate were shown to be potent inhibitors of the reduction reaction. Thus, compounds of the N-alkyl-8-aza-4 alpha,10-dimethyl-trans-decal-3 beta-ol series strongly inhibited sterol reductase (I50 = 0.07 - 4 microM) (I50/Km = 10(-4) - 10(-3), as did the antimycotic agent 15-azasterol (I50 = 0.03 microM); all of these compounds act as reaction-intermediate analogues of the proposed C14 carbonium ion intermediate. Moreover, the in vitro inhibition of the plant sterol reductase by a series of ammonium-ion-containing fungicides was demonstrated. The relative specificity of these different series of inhibitors toward cycloeucalenol-obtusifoliol isomerase, delta 8----delta 7-sterol isomerase and delta 8,14-sterol delta 14-reductase, was directly studied.     

Inhibition of δ8 → δ7-sterol isomerase and of cycloeucalenol—obtusifoliol isomerase by N-benzyl-8-aza-4α, 10-dimethyl-trans-decal-3β-ol,an analogue of a carbocationic high energy intermediate

An enzymatic assay for the δ⁸ → δ⁷-sterol isomerase, an enzyme involved in sterol biosynthesis, has been developed in higher plants. This assay has been used in the study of various inhibitors. N-Benzyl-8-aza-4α, 10-dimethyl- trans-decal-3β-ol was designed to mimic the C-8 and the C-9 carbocationic high energy intermediates occurring during the reactions catalysed by the δ⁸ → δ⁷-sterol isomerase and the cycloeucalenol obtusifoliol isomerase, respectively. In accordance with the ‘transition state analogues’ theory, this analogue of a high energy intermediate was found to be a very potent and specific inhibitor of the two enzymatic reactions both in vitro and in vivo.     

Comparison of the subsite specificity of the mammalian neutral endopeptidase 24.11 (enkephalinase) to the bacterial neutral endopeptidase thermolysin

A comparison has been made of the specificity of the mammalian neutral metalloendopeptidase, endopeptidase 24.11, with that of the bacterial neutral metalloendopeptidase thermolysin. A series of synthetic oligopeptides which have previously been studied as substrates for thermolysin and used in computer modeling were examined as substrates for the mammalian enzyme. It was found that P1, P2, and P'3 subsite interactions in the mammalian enzyme, although similar to those found in thermolysin, are less restrictive spatially and are considerably less dependent on hydrophobic interactions. This difference was maximally expressed with the synthetic substrate dansyl-D-alanylglycylnitrophenylalanylglycine which is a substrate for the mammalian enzyme, but not for the bacterial enzyme. A comparison of substrates in the free acid form with their corresponding amides showed that binding to the mammalian enzyme is dependent in part on an ionic interaction between the substrate carboxylate group and the enzyme. Such an ionic interaction was not observed with the bacterial enzyme.     

Circular dichroic evidence for an ordered sequence of ligand/binding site interactions in the catalytic reaction of the cAMP-dependent protein kinase

A limiting requirement for substrate specificity of the cAMP-dependent protein kinase is the presence of one or two basic residues located to the N-terminal side of the target substrate serine. Furthermore, circular dichroic (CD) studies have shown that binding of protein substrate involves a series of at least two independent conformational changes in the enzyme, each of which is initiated by a recognition signal on the substrate protein. The present study attempts to elucidate further the complete sequence of enzyme/ligand interactions by using the synthetic substrate peptide Kemptide and analogues differing from it at crucial points in the sequence: the Ala-peptide, where alanine is substituted for the target serine, and D-Ser-Kemptide, where the target serine is in the D rather than the L configuration. Examination of the effects of binding of these substrates on the intrinsic UV CD of the enzyme and the induced CD in the presence of Blue Dextran has revealed a third step in the substrate/enzyme binding interaction. Although sections of the conformational change at the active site are dependent on the basic subsite and the serine hydroxyl group on the peptide, respectively, the complete conformational change requires that the substrate be bound in random coil conformation. Where this does not occur, the kinetics show that the peptide will not act either as substrate or as inhibitor of the enzyme. Further, the interaction between the serine hydroxyl group and an enzyme tyrosine residue, previously observed, appears to be dependent on the correct orientation as well as the mere presence of the target -OH group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and evaluation of non-hydrolyzable d-mannose 6-phosphate surrogates reveal 6-deoxy-6-dicarboxymethyl-d-mannose as a new strong inhibitor of phosphomannose isomerases

Non-hydrolyzable d-mannose 6-phosphate analogues in which the phosphate group was replaced by a phosphonomethyl, a dicarboxymethyl, or a carboxymethyl group were synthesized and kinetically evaluated as substrate analogues acting as potential inhibitors of type I phosphomannose isomerases (PMIs) from Saccharomyces cerevisiae and Escherichia coli. While 6-deoxy-6-phosphonomethyl-d-mannose and 6-deoxy-6-carboxymethyl-d-mannose did not inhibit the enzymes significantly, 6-deoxy-6-dicarboxymethyl-d-mannose appeared as a new strong competitive inhibitor of both S. cerevisiae and E. coli PMIs with K_m/K_i ratios of 28 and 8, respectively. We thus report the first malonate-based inhibitor of an aldose–ketose isomerase to date. Phosphonomethyl mimics of the 1,2-cis-enediolate high-energy intermediate postulated for the isomerization reaction catalyzed by PMIs were also synthesized but behave as poor inhibitors of PMIs. A polarizable molecular mechanics (SIBFA) study was performed on the complexes of d-mannose 6-phosphate and two of its analogues with PMI from Candida albicans, an enzyme involved in yeast infection homologous to S. cerevisiae and E. coli PMIs. It shows that effective binding to the catalytic site occurs with retention of the Zn(II)-bound water molecule. Thus the binding of the hydroxyl group on C1 of the ligand to Zn(II) should be water-mediated. The kinetic study reported here also suggests the dianionic character of the phosphate surrogate as a likely essential parameter for strong binding of the inhibitor to the enzyme active site.     

7-Oxo-24ξ(28)-dihydrocycloeucalenol,a potent inhibitor of plant sterol biosynthesis

Cycloeucalenol-obtusifoliol isomerase from higher plant cells catalyses the opening of the cyclopropane ring of cycloeucalenol yielding obtusifoliol. 7-Oxo-24ξ(28)-dihydrocycloeucalenol was not a substrate but behaved like a potent inhibitor of the isomerase. The inhibition was reversible and highly specific; the inhibitor needed the presence of the 7-oxo group, the cyclopropane ring and the absence of a 4β-methyl group to be active. Other enzymes involved in plant sterol biosynthesis such as 2, 3-oxidosqualene-cycloartenol cyclase and S-adenosyl methionine cycloartenol C-24 methyltransferase were not inhibited by 7-oxo-24ξ(28)-dihydrocycloeucalenol. In vivo treatment of a suspension of bramble cells growing in a liquid medium with 7-oxo-24ξ(28)-dihydrocycloeucalenol resulted in a strong accumulation of 9β 19-cyclopropyl sterols confirming that the main cellular target of the inhibitor is the cycloeucalenol-obtusifoliol isomerase.     

Depolymerisation of F-actin to G-actin and its repolymerisation in the presence of analogs of adenosine triphosphate.

The binding of the coenzyme to octopine dehydrogenase was investigated by kinetic and spectroscopic studies using different analogues of NAD+. The analogues employed were fragments of the coenzyme molecule and dinucleotides modified on the purine or the pyridine ring. The binding of ADPribose is sufficient to induce local conformational changes necessary for the good positioning of substrates. AMP, ADP, NMN+ and NMNH do not show this effect. Analogues modified on the purine ring such as nicotinamide deaminoadenine dinucleotide, nicotinamide--8-bromoadenine dinucleotide, nicotinamide--8-thioadenine dinucleotide and nicotinamide 1: N6-ethenoadenine dinucleotide bind to the enzyme and give catalytically active ternary complexes. Modifications of the pyridine ring show an important effect on the binding of the coenzyme as well as on the formation of ternary complexes. Thus, the carboxamide group can well be replaced by an acetyl group and also, though less efficiently, by a formyl or cyano group. However more bulky substituents such as thio, chloroacetyl or propionyl groups prevent the binding. The analogues bearing a methyl group in the 4 or 5 position, which are competitive inhibitors, are able to give binary by not ternary complexes. The case of 1,4,5,6-tetrahydronicotinamide--adenine dinucleotide which does not give ternary complexes like NADH is discussed. The above findings show that the pyridine and adenine parts are both involved in the binding of the coenzyme and of the substrate to octopine dehydrogenase. The nicotinamide binding site of this enzyme seems to be the most specific and restricted one among the dehydrogenases so far described. The protective effects of coenzyme analogues towards essential -SH group were also studied.     

Determinants of substrate specificity for saccharopine dehydrogenase from Saccharomyces cerevisiae

A survey of NADH, alpha-Kg, and lysine analogues has been undertaken in an attempt to define the substrate specificity of saccharopine dehydrogenase and to identify functional groups on all substrates and dinucleotides important for substrate binding. A number of NAD analogues, including NADP, 3-acetylpyridine adenine dinucleotide (3-APAD), 3-pyridinealdehyde adenine dinucleotide (3-PAAD), and thionicotinamide adenine dinucleotide (thio-NAD), can serve as a substrate in the oxidative deamination reaction, as can a number of alpha-keto analogues, including glyoxylate, pyruvate, alpha-ketobutyrate, alpha-ketovalerate, alpha-ketomalonate, and alpha-ketoadipate. Inhibition studies using nucleotide analogues suggest that the majority of the binding energy of the dinucleotides comes from the AMP portion and that distinctly different conformations are generated upon binding of the oxidized and reduced dinucleotides. Addition of the 2'-phosphate as in NADPH causes poor binding of subsequent substrates but has little effect on coenzyme binding and catalysis. In addition, the 10-fold decrease in affinity of 3-APAD in comparison to NAD suggests that the nicotinamide ring binding pocket is hydrophilic. Extensive inhibition studies using aliphatic and aromatic keto acid analogues have been carried out to gain insight into the keto acid binding pocket. Data suggest that a side chain with three carbons (from the alpha-keto group up to and including the side chain carboxylate) is optimal. In addition, the distance between the C1-C2 unit and the C5 carboxylate of the alpha-keto acid is also important for binding; the alpha-oxo group contributes a factor of 10 to affinity. The keto acid binding pocket is relatively large and flexible and can accommodate the bulky aromatic ring of a pyridine dicarboxylic acid and a negative charge at the C3 but not the C4 position. However, the amino acid binding site is hydrophobic, and the optimal length of the hydrophobic portion of the amino acid carbon side chain is three or four carbons. In addition, the amino acid binding pocket can accommodate a branch at the gamma-carbon, but not at the beta-carbon.     

Substrate specificity of mammalian folylpoly-gamma-glutamate synthetase for 5,8-dideazafolates and 5,8-dideaza analogues of aminopterin

The substrate specificity of pig liver folylpolyglutamate synthetase (tetrahydrofolate:L-glutamate gamma-ligase (ADP-forming), EC 6.3.2.17) for classical 5,8-dideaza analogues of folic acid, isofolic acid aminopterin and isoaminopterin has been investigated. 5,8-Dideazafolate and 5,8-dideazaaminopterin are very effective substrates with activities approaching those of the best reduced folate substrates. The analogous isofolate analogues are less effective substrates, but still better than folic acid. The 5-chloro substituent is the only modification that consistently increases the on rate, with 5-chloro-5,8-dideazaaminopterin being the most effective substrate found, thus far, for the enzyme. Methylation at positions 9 or 10 generally decreases binding, while 5-methylation increases the binding of 4-oxoquinazolines, but decreases the binding of their 4-amino counterparts. The presence of a formyl group at N9 or N10 has the opposite effect, decreasing the binding of 4-oxo analogues while increasing the rate for 4-amino derivatives. Increases in on rate with methyl, formyl or 4-amino substitutions are only significant when the parent compound is a poor substrate, suggesting that these groups do not interact directly with the enzyme but cause conformational changes in the structure of the substrate that influence binding to the enzyme.     

Esters of mandelic acid as substrates for (S)-mandelate dehydrogenase from Pseudomonas putida: implications for the reaction mechanism

(S)-Mandelate dehydrogenase (MDH) from Pseudomonas putida is a flavin mononucleotide (FMN)-dependent enzyme that oxidizes (S)-mandelate to benzoylformate. In this work, we show that the ethyl and methyl esters of (S)-mandelic acid are substrates for MDH. Although the binding affinity of the neutral esters is 25-50-fold lower relative to the negatively charged (S)-mandelate, they are oxidized with comparable k(cat)s. Substrate analogues in which the carbonyl group on the C-1 carbon is replaced by other electron-withdrawing groups were not substrates. The requirement of a carbonyl group on the C-1 carbon in a substrate suggests that the negative charge developed during the reaction is stabilized by delocalization to the carbonyl oxygen. Arg277, a residue that is important in both binding and transition state stabilization for the activity with (S)-mandelate, is also critical for transition state stabilization for the esters, but not for their binding affinity. We previously showed that the substrate oxidation half-reaction with (S)-mandelate has two rate-limiting steps of similar activation energies and proceeds through the formation of a charge-transfer complex of an electron-rich donor and oxidized FMN [Dewanti, A. R., and Mitra, B. (2003) Biochemistry 42, 12893-12901]. This charge-transfer intermediate was observed with the neutral esters as well. The observation of this electron-rich intermediate for the oxidation of an uncharged substrate to an uncharged product, as well as the critical role of Arg277 in the reaction with the esters, provides further evidence that the MDH reaction mechanism is not a concerted transfer of a hydride ion from the substrate to the FMN, but involves the transient formation of a carbanion/ene(di)olate intermediate.     

Distinct binding of cholesterol and 5beta-cholestane-3alpha,7alpha,12alpha-triol to cytochrome P450 27A1: evidence from modeling and site-directed mutagenesis studies

Cytochrome P450 27A1 (P450 27A1 or CYP27A1) is an important enzyme that participates in different pathways of cholesterol degradation as well as in the activation of vitamin D(3). Several approaches were utilized to investigate how two physiological substrates, cholesterol and 5beta-cholestane-3alpha,7alpha,12alpha-triol, interact with CYP27A1. The enzyme active site was first probed spectrally by assessing binding of the two substrates and five substrate analogues followed by computer modeling and site-directed mutagenesis. The computer models suggest that the spatial positions and orientations of cholesterol and 5beta-cholestane-3alpha,7alpha,12alpha-triol are different in the enzyme active site. As a result, some of the active site residues interact with both substrates, although they are situated differently relative to each steroid, and some residues bind only one substrate. Mutation of the overlapping substrate-contact residues (W100, H103, T110, M301C, V367, I481, and V482) affected CYP27A1 binding and enzyme activity in a substrate-dependent manner and allowed identification of several important side chains. T110 is proposed to interact with the 12alpha-hydroxyl of 5beta-cholestane-3alpha,7alpha,12alpha-triol, whereas V367 seems to be crucial for correct positioning of the cholesterol C26 methyl group and for regioselective hydroxylation of this substrate. Distinct binding of the CYP27A1 substrates may provide insight into why phenotypic manifestations of cerebrotendinous xanthomatosis, a disease associated with CYP27A1 deficiency, are so diverse.     

Structure of mandelate racemase with bound intermediate analogues benzohydroxamate and cupferron

Mandelate racemase (MR, EC 5.1.2.2) from Pseudomonas putida catalyzes the Mg(2+)-dependent interconversion of the enantiomers of mandelate, stabilizing the altered substrate in the transition state by 26 kcal/mol relative to the substrate in the ground state. To understand the origins of this binding discrimination, we determined the X-ray crystal structures of wild-type MR complexed with two analogues of the putative aci-carboxylate intermediate, benzohydroxamate and Cupferron, to 2.2-? resolution. Benzohydroxamate is shown to be a reasonable mimic of the transition state and/or intermediate because its binding affinity for 21 MR variants correlates well with changes in the free energy of transition state stabilization afforded by these variants. Both benzohydroxamate and Cupferron chelate the active site divalent metal ion and are bound in a conformation with the phenyl ring coplanar with the hydroxamate and diazeniumdiolate moieties, respectively. Structural overlays of MR complexed with benzohydroxamate, Cupferron, and the ground state analogue (S)-atrolactate reveal that the para carbon of the substrate phenyl ring moves by 0.8-1.2 ? between the ground state and intermediate state, consistent with the proposal that the phenyl ring moves during MR catalysis while the polar groups remain relatively fixed. Although the overall protein structure of MR with bound intermediate analogues is very similar to that of MR with bound (S)-atrolactate, the intermediate-Mg(2+) distance becomes shorter, suggesting a tighter complex with the catalytic Mg(2+). In addition, Tyr 54 moves closer to the phenyl ring of the bound intermediate analogues, contributing to an overall constriction of the active site cavity. However, site-directed mutagenesis experiments revealed that the role of Tyr 54 in MR catalysis is relatively minor, suggesting that alterations in enzyme structure that contribute to discrimination between the altered substrate in the transition state and the ground state by this proficient enzyme are extremely subtle.     

Triosephosphate isomerase: removal of a putatively electrophilic histidine residue results in a subtle change in catalytic mechanism

An important active-site residue in the glycolytic enzyme triosephosphate isomerase is His-95, which appears to act as an electrophilic component in catalyzing the enolization of the substrates. With the techniques of site-directed mutagenesis, His-95 has been replaced by Gln in the isomerase from Saccharomyces cerevisiae. The mutant isomerase has been expressed in Escherichia coli strain DF502 and purified to homogeneity. The specific catalytic activity of the mutant enzyme is less than that of wild type by a factor of nearly 400. The mutant enzyme can be resolved from the wild-type isomerase on nondenaturing isoelectric focusing gels, and an isomerase activity stain shows that the observed catalytic activity indeed derives from the mutant protein. The inhibition constants for arsenate and for glycerol phosphate with the mutant enzyme are similar to those with the wild-type isomerase, but the substrate analogues 2-phosphoglycolate and phosphoglycolohydroxamate bind 8- and 35-fold, respectively, more weakly to the mutant isomerase. The mutant enzyme shows the same stereospecificity of proton transfer as the wild type. Tritium exchange experiments similar to those used to define the free energy profile for the wild-type yeast isomerase, together with a new method of analysis involving 14C and 3H doubly labeled substrates, have been used to investigate the energetics of the mutant enzyme catalyzed reaction. When the enzymatic reaction is conducted in tritiated solvent, the mutant isomerase does not catalyze any appreciable exchange between protons of the remaining substrate and those of the solvent either in the forward reaction direction (using dihydroxyacetone phosphate as substrate) or in the reverse direction (using glyceraldehyde phosphate as substrate). However, the specific radioactivity of the product glyceraldehyde phosphate formed in the forward reaction is 31% that of the solvent, while that of the product dihydroxyacetone phosphate formed in the reverse reaction is 24% that of the solvent. The deuterium kinetic isotope effects observed with the mutant isomerase using [1(R)-2H]dihydroxyacetone phosphate and [2-2H]glyceraldehyde 3-phosphate are 2.15 +/- 0.04 and 2.4 +/- 0.1, respectively. These results lead to the conclusion that substitution of Gln for His-95 so impairs the ability of the enzyme to stabilize the reaction intermediate that there is a change in the pathways of proton transfer mediated by the mutant enzyme. The data allow us more closely to define the role of His-95 in the reaction catalyzed by the wild-type enzyme, while forcing us to be alert to subtle changes in mechanistic pathways when mutant enzymes are generated.     

Role of the charged groups of glutathione disulfide in the catalysis of glutathione reductase: crystallographic and kinetic studies with synthetic analogues

Six analogues of glutathione disulfide were synthesized. All of them involved the abolishment of charges, either by amidation of carboxylates or by removal of amino groups. Four of these analogues could be bound to crystalline oxidized glutathione reductase, and their binding modes could be established by X-ray analyses at 2.4-A resolution. All six analogues were catalytically processed; the kinetic parameters were determined. The two analogues that did not bind in the crystals had by far the poorest catalytic efficiencies. Kinetic parameters together with X-ray data show the influence of each charged group on binding and catalytic rate. Data analysis indicates that the enzyme avoids processing of incorrect substrates in two ways: First, it reduces their binding strengths and/or enforces displacement of catalytically important substrate parts. Furthermore, it forms a fragile cluster of bound substrate and catalytically competent residues, which is unbalanced by incorrect parts of the substrate such that catalysis is prevented. A scouting microcalorimetric study using glutathione disulfide yielded a binding enthalpy of -103 (+/- 10) kJ/mol at 25 degrees C and a heat capacity change of -8 (+/- 1) kJ.mol-1.K-1. The study showed that it is feasible to measure these parameters as a function of substrate modification.     

Reversal of enzyme regiospecificity with alternative substrates for aspartokinase I from Escherichia coli.

The substrate specificity of aspartokinase I has been examined by using both steady-state kinetic analyses and phosphorus-31 NMR spectroscopic studies. Analogues in which the alpha-amino group is either derivatized or replaced are not substrates or inhibitors for the enzyme, indicating the importance of the alpha-amino group as a binding determinant. The alpha-carboxyl group is not required for substrate recognition, and the alpha-amide or alpha-esters are competent alternative substrates. In addition, beta-derivatized structural analogues, such as the beta-hydroxamate, the beta-amide, or beta-esters, were found to be viable substrates. This was unexpected since the beta-carboxyl group is the usual site of phosphorylation. The nature of the acyl phosphate products obtained from these beta-derivatized alternative substrates has been characterized by coupled enzyme assays, oxygen-18-labeling studies, and phosphorus-31 NMR spectroscopy. These beta-derivatized analogues are capable of productive binding to aspartokinase through a reversal of regiospecificity to make the alpha-carboxyl group available as a phosphoryl acceptor. Many, but not all, of these alpha-acyl phosphates have also been shown to be viable substrates for the next two enzyme-catalyzed steps in this metabolic pathway. This raises the possibility of producing enzyme-generated alternative substrates that can serve as antimetabolites for the downstream reactions in this biosynthetic pathway.     

Enzymatic incorporation of ATP and CTP analogues into the 3' end of tRNA

Structural analogues of adenosine 5'-triphosphate and cytidine 5'-triphosphate were investigated as substrates for ATP(CTP):tRNA nucleotidyl transferase. Eight out of 26 ATP analogues and six out of nine CTP analogues were incorporated into the 3' terminus of tRNA. In general, for the recognition of the substrates the modification of the cytidine is less critical than is the modification of adenosine. An isosteric substitution on the ribose residue is possible in both CTP and ATP. The free hydroxyls of these triphosphates can be replaced by an amino group or hydrogen atom without loss of substrate properties. Modifications of positions 1, 2, 6, and 8 on the adenine ring of ATP are not allowed whereas modification on positions 2, 4 and 5 on the cytosine ring of CTP are tolerated by the enzyme. No differences can be observed in the substrate properties of ATP(CTP):tRNA nucleotidyl transferase isolated from different sources. Methods for preparation of tRNA species, which are shortened at their 3' end by one or more nucleotides, and analytical procedures for characterisation of these modified tRNAs are described.     

Crystal structure of rabbit phosphoglucose isomerase complexed with its substrate D-fructose 6-phosphate.

Phosphoglucose isomerase (PGI, EC 5.3.1.9) catalyzes the interconversion of D-glucose 6-phosphate (G6P) and D-fructose 6-phosphate (F6P) and plays important roles in glycolysis and gluconeogenesis. Biochemical characterization of the enzyme has led to a proposed multistep catalytic mechanism. First, the enzyme catalyzes ring opening to yield the open chain form of the substrate. Then isomerization proceeds via proton transfer between C2 and C1 of a cis-enediol(ate) intermediate to yield the open chain form of the product. Catalysis proceeds in both the G6P to F6P and F6P to G6P directions, so both G6P and F6P are substrates. X-ray crystal structure analysis of rabbit and bacterial PGI has previously identified the location of the enzyme active site, and a recent crystal structure of rabbit PGI identified Glu357 as a candidate functional group for transferring the proton. However, it was not clear which active site amino acid residues catalyze the ring opening step. In this paper, we report the X-ray crystal structure of rabbit PGI complexed with the cyclic form of its substrate, D-fructose 6-phosphate, at 2.1 A resolution. The location of the substrate relative to the side chains of His388 suggest that His388 promotes ring opening by protonating the ring oxygen. Glu216 helps to position His388, and a water molecule that is held in position by Lys518 and Thr214 accepts a proton from the hydroxyl group at C2. Comparison to a structure of rabbit PGI with 5PAA bound indicates that ring opening is followed by loss of the protonated water molecule and conformational changes in the substrate and the protein so that a helix containing amino acids 513-520 moves in toward the substrate to form additional hydrogen bonds with the substrate.     

Intrinsic enoyl-CoA isomerase activity of rat acyl-CoA oxidase I

Rat peroxisomal acyl-CoA oxidase I is a key enzyme for the beta-oxidation of fatty acids, and the deficiency of this enzyme in patient has been previously reported. It was found that rat acyl-CoA oxidase I has intrinsic enoyl-CoA isomerase activity, which was confirmed using incubation followed with HPLC analysis in this study. Various 3-enoyl-CoA substrates with cis or trans configuration were synthesized and used in the study of enzyme substrate specificity. The isomerase activity of the enzyme was characterized through studies of kinetics, pH dependence, and enzyme inhibition. Most k(cat)/K(M) values of rat peroxisomal acyl-CoA oxidase I for isomerization reaction are comparable with those of authentic rat liver peroxisomal Delta(3)-Delta(2)-enoyl-CoA isomerase and rat liver peroxisomal multifunctional enzyme 1 when hexenoyl-CoA and octenoyl-CoA with cis- or trans-configuration were used as substrate. Glu421 was found to be the catalytic residue for both oxidase and isomerase activities of the enzyme. The isomerase activity of rat peroxisomal acyl-CoA oxidase I is probably due to a spontaneous process driven by thermodynamic equilibrium with formation of a conjugated structure after deprotonation of substrate alpha-proton. The energy level of transition state may be lowered by a stable dienolate intermediate, which gain further stabilization via charge transfer with electron-deficient FAD cofactor of the enzyme.     

Anomeric specificity of D-xylose isomerase.

Crystal structures of complexes of D-xylose isomerase with deoxysugars have been determined. Deoxynojirimycin is a structural analogue of alpha-pyranose and mimics the binding of these aldose substrates. The structure of this complex supports the hypothesis that an imidazole group catalyzes ring opening of the pyranose. The steric restrictions in the active site of the enzyme prevent a beta-pyranose from binding in the same way. For the reverse reaction with ketoses, the anomeric specificity is less certain. Dideoxyimino-D-glucitol is a structural analogue of the ketose alpha-D-furanose. The binding of the inhibitor dideoxyimino-D-glucitol to the crystals of the enzyme does not mimic the binding of the reactive alpha-D-fructofuranose. Superposition of the nonphysiological substrate alpha-D-fructofuranose onto the atomic positions of dideoxyimino-D-glucitol is not possible due to the steric restrictions of the active site. However, by utilizing the approximate 2-fold symmetry of the sugar, a stereochemically sensible model is produced which is consistent with other data. In addition to reaction with alpha-D-furanose, the enzyme probably reacts with open ring keto sugars which are present at significant concentrations. Other sugars which resemble furanoses either do not inhibit significantly or are not observed in the crystals bound in a single conformation.