Cation mediation of radical transfer between Trp48 and Tyr356 during O2 activation by protein R2 of Escherichia coli ribonucleotide reductase: relevance to R1-R2 radical transfer in nucleotide reduction?

A tryptophan 48 cation radical (W48(+)(*)) forms concomitantly with the Fe(2)(III/IV) cluster, X, during activation of oxygen for tyrosyl radical (Y122.) production in the R2 subunit of class I ribonucleotide reductase (RNR) from Escherichia coli. W48(+)(*) is also likely to be an intermediate in the long-range radical transfer between R2 and its partner subunit, R1, during nucleotide reduction by the RNR holoenzyme. The kinetics of decay of W48(+)(*) and formation of tyrosyl radicals during O(2) activation (in the absence of R1) in wild-type (wt) R2 and in variants with either Y122, Y356 (the residue thought to propagate the radical from W48(+)(*) into R1 during turnover), or both replaced by phenylalanine (F) have revealed that the presence of divalent cations at concentrations similar to the [Mg(2+)] employed in the standard RNR assay (15 mM) mediates a rapid radical-transfer equilibrium between W48 and Y356. Cation-mediated propagation of the radical from W48 to Y356 gives rise to a fast phase of Y. production that is essentially coincident with W48(+)(*) formation and creates an efficient pathway for decay of W48(+)(*). Possible mechanisms of this cation mediation and its potential relevance to intersubunit radical transfer during nucleotide reduction are considered.     

Generation of the R2 subunit of ribonucleotide reductase by intein chemistry: insertion of 3-nitrotyrosine at residue 356 as a probe of the radical initiation process

Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleoside diphosphates. The enzyme is composed of two subunits: R1 and R2. R1 contains the active site for nucleotide reduction and the allosteric effector sites that regulate the specificity and turnover rate. R2 contains the diferric-tyrosyl (Y(*)) radical cofactor that initiates nucleotide reduction by a putative long-range proton-coupled electron transfer (PCET) pathway over 35 A. This pathway is thought to involve specific amino acid radical intermediates (Y122 to W48 to Y356 within R2 to Y731 to Y730 to C439 within R1). In an effort to study radical initiation, R2 (375 residues) has been synthesized semisynthetically. R2 (residues 1-353), attached to an intein and a chitin binding domain, was constructed, and the protein was expressed (construct 1). This construct was then incubated with Fe(2+) and O(2) to generate the diferric-Y(*) cofactor, and the resulting protein was purified using a chitin affinity column. Incubation of construct 1 with 2-mercaptoethanesulfonic acid (MESNA) resulted in the MESNA thioester of R2 (1-353) (construct 2). A peptide containing residues 354-375 of R2 was generated using solid-phase peptide synthesis where 354, a serine in the wild-type (wt) R2, was replaced by a cysteine. Construct 2 and this peptide were ligated, and the resulting full-length R2 was separated from truncated R2 by anion-exchange chromatography. The purified protein had a specific activity of 350 nmol min(-1) mg(-1), identical to the same protein generated by site-directed mutagenesis when normalized for Y(*). As a first step in studying the radical initiation by PCET, R2 was synthesized with Y356 replaced by 3-nitrotyrosine (NO(2)Y). The protein is inactive (specific activity 1 x 10(-4) that of wt-R2), which permitted a determination of the pK(a) of the NO(2)Y in the R1/R2 complex in the presence of substrate and effectors. Under all conditions, the pK(a) was minimally perturbed. This has important mechanistic implications for the radical initiation process.     

Use of 2,3,5-F(3)Y-β2 and 3-NH(2)Y-α2 to study proton-coupled electron transfer in Escherichia coli ribonucleotide reductase

Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleoside 5'-diphosphates (NDPs) to deoxynucleotides (dNDPs). The active site for NDP reduction resides in α2, and the essential diferric-tyrosyl radical (Y(122)(?)) cofactor that initiates transfer of the radical to the active site cysteine in α2 (C(439)), 35 ? removed, is in β2. The oxidation is proposed to involve a hopping mechanism through aromatic amino acids (Y(122) → W(48) → Y(356) in β2 to Y(731) → Y(730) → C(439) in α2) and reversible proton-coupled electron transfer (PCET). Recently, 2,3,5-F(3)Y (F(3)Y) was site-specifically incorporated in place of Y(356) in β2 and 3-NH(2)Y (NH(2)Y) in place of Y(731) and Y(730) in α2. A pH-rate profile with F(3)Y(356)-β2 suggested that as the pH is elevated, the rate-determining step of RNR can be altered from a conformational change to PCET and that the altered driving force for F(3)Y oxidation, by residues adjacent to it in the pathway, is responsible for this change. Studies with NH(2)Y(731(730))-α2, β2, CDP, and ATP resulted in detection of NH(2)Y radical (NH(2)Y(?)) intermediates capable of dNDP formation. In this study, the reaction of F(3)Y(356)-β2, α2, CDP, and ATP has been examined by stopped-flow (SF) absorption and rapid freeze quench electron paramagnetic resonance spectroscopy and has failed to reveal any radical intermediates. The reaction of F(3)Y(356)-β2, CDP, and ATP has also been examined with NH(2)Y(731)-α2 (or NH(2)Y(730)-α2) by SF kinetics from pH 6.5 to 9.2 and exhibited rate constants for NH(2)Y(?) formation that support a change in the rate-limiting step at elevated pH. The results together with kinetic simulations provide a guide for future studies to detect radical intermediates in the pathway.     

Studies on ribonucleoside-diphosphate reductase from Escherichia coli. The product dCDP is a competitive inhibitor and functions as a spectroscopic probe for the substrate binding site; demonstration by enzyme kinetics and 1H NMR.

Ribonucleoside-diphosphate reductase (EC 1.17.4.1) from Escherichia coli consists of two protein subunits, R1 of 171.5 kDa and R2 of 86.8 kDa, and catalyzes the reduction of all four common ribonucleoside diphosphates. In a search for ligands that bind weakly to the enzyme active site and may be in fast exchange suitable for NMR studies, we have found that the product dCDP is a competitive inhibitor. Kinetics with CDP as substrate shows Km = 4.8 x 10(-5) M and dCDP inhibits with Ki = 1.6 x 10(-4) M. With an assumed diffusion limited binding rate approximately less than 10(9) M-1s-1, the dissociation rate of dCDP would be approximately less than 10(5) s-1. In 1H-NMR experiments studying linewidths, i.e. spin-spin relaxation, dCDP is indeed demonstrated to be in fast exchange. Enzyme subunit R1 causes a line broadening of dCDP resonances. Unexpectedly less broadening was observed when subunit R2 combined with R1. No paramagnetic interaction from the tyrosyl radical of R2 could be detected. It is concluded that dCDP is a promising NMR probe for studies of active-site properties of the enzyme.     

Mechanism of DNA polymerization catalyzed by Sulfolobus solfataricus P2 DNA polymerase IV

The kinetic mechanism of DNA polymerization catalyzed by Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) is resolved by pre-steady-state kinetic analysis of single-nucleotide (dTTP) incorporation into a DNA 21/41-mer. Like replicative DNA polymerases, Dpo4 utilizes an "induced-fit" mechanism to select correct incoming nucleotides. The affinity of DNA and a matched incoming nucleotide for Dpo4 was measured to be 10.6 nM and 230 microM, respectively. Dpo4 binds DNA with an affinity similar to that of replicative polymerases due to the presence of an atypical little finger domain and a highly charged tether that links this novel domain to its small thumb domain. On the basis of the elemental effect between the incorporations of dTTP and its thio analogue S(p)-dTTPalphaS, the incorporation of a correct incoming nucleotide by Dpo4 was shown to be limited by the protein conformational change step preceding the chemistry step. In contrast, the chemistry step limited the incorporation of an incorrect nucleotide. The measured dissociation rates of the enzyme.DNA binary complex (0.02-0.07 s(-1)), the enzyme.DNA.dNTP ternary complex (0.41 s(-1)), and the ternary complex after the protein conformational change (0.004 s(-1)) are significantly different and support the existence of a bona fide protein conformational change step. The rate-limiting protein conformational change was further substantiated by the observation of different reaction amplitudes between pulse-quench and pulse-chase experiments. Additionally, the processivity of Dpo4 was calculated to be 16 at 37 degrees C from analysis of a processive polymerization experiment. The structural basis for both the protein conformational change and the low processivity of Dpo4 was discussed.     

Transient-state and steady-state kinetic studies of the mechanism of NADH-dependent aldehyde reduction catalyzed by xylose reductase from the yeast Candida tenuis

Microbial xylose reductase, a representative aldo-keto reductase of primary sugar metabolism, catalyzes the NAD(P)H-dependent reduction of D-xylose with a turnover number approximately 100 times that of human aldose reductase for the same reaction. To determine the mechanistic basis for that physiologically relevant difference and pinpoint features that are unique to the microbial enzyme among other aldo/keto reductases, we carried out stopped-flow studies with wild-type xylose reductase from the yeast Candida tenuis. Analysis of transient kinetic data for binding of NAD(+) and NADH, and reduction of D-xylose and oxidation of xylitol at pH 7.0 and 25 degrees C provided estimates of rate constants for the following mechanism: E + NADH right arrow over left arrow E.NADH right arrow over left arrow E.NADH + D-xylose right arrow over left arrow E.NADH.D-xylose right arrow over left arrow E.NAD(+).xylitol right arrow over left arrow E.NAD(+) right arrow over left arrow E.NAD(+) right arrow over left arrow E + NAD(+). The net rate constant of dissociation of NAD(+) is approximately 90% rate limiting for k(cat) of D-xylose reduction. It is controlled by the conformational change which precedes nucleotide release and whose rate constant of 40 s(-)(1) is 200 times that of completely rate-limiting E.NADP(+) --> E.NADP(+) step in aldehyde reduction catalyzed by human aldose reductase [Grimshaw, C. E., et al. (1995) Biochemistry 34, 14356-14365]. Hydride transfer from NADH occurs with a rate constant of approximately 170 s(-1). In reverse reaction, the E.NADH --> E.NADH step takes place with a rate constant of 15 s(-1), and the rate constant of ternary-complex interconversion (3.8 s(-1)) largely determines xylitol turnover (0.9 s(-1)). The bound-state equilibrium constant for C. tenuis xylose reductase is estimated to be approximately 45 (=170/3.8), thus greatly favoring aldehyde reduction. Formation of productive complexes, E.NAD(+) and E.NADH, leads to a 7- and 9-fold decrease of dissociation constants of initial binary complexes, respectively, demonstrating that 12-fold differential binding of NADH (K(i) = 16 microM) vs NAD(+) (K(i) = 195 microM) chiefly reflects difference in stabilities of E.NADH and E.NAD(+). Primary deuterium isotope effects on k(cat) and k(cat)/K(xylose) were, respectively, 1.55 +/- 0.09 and 2.09 +/- 0.31 in H(2)O, and 1.26 +/- 0.06 and 1.58 +/- 0.17 in D(2)O. No deuterium solvent isotope effect on k(cat)/K(xylose) was observed. When deuteration of coenzyme selectively slowed the hydride transfer step, (D)()2(O)(k(cat)/K(xylose)) was inverse (0.89 +/- 0.14). The isotope effect data suggest a chemical mechanism of carbonyl reduction by xylose reductase in which transfer of hydride ion is a partially rate-limiting step and precedes the proton-transfer step.     

In vivo assay for low-activity mutant forms of Escherichia coli ribonucleotide reductase

Ribonucleotide reductase (RNR) catalyzes the essential production of deoxyribonucleotides in all living cells. In this study we have established a sensitive in vivo assay to study the activity of RNR in aerobic Escherichia coli cells. The method is based on the complementation of a chromosomally encoded nonfunctional RNR with plasmid-encoded RNR. This assay can be used to determine in vivo activity of RNR mutants with activities beyond the detection limits of traditional in vitro assays. E. coli RNR is composed of two homodimeric proteins, R1 and R2. The R2 protein contains a stable tyrosyl radical essential for the catalysis that takes place at the R1 active site. The three-dimensional structures of both proteins, phylogenetic studies, and site-directed mutagenesis experiments show that the radical is transferred from the R2 protein to the active site in the R1 protein via a radical transfer pathway composed of at least nine conserved amino acid residues. Using the new assay we determined the in vivo activity of mutants affecting the radical transfer pathway in RNR and identified some residual radical transfer activity in two mutant R2 constructs (D237N and W48Y) that had previously been classified as negative for enzyme activity. In addition, we show that the R2 mutant Y356W is completely inactive, in sharp contrast to what has previously been observed for the corresponding mutation in the mouse R2 enzyme.     

Kinetic characterization of the ATPase cycle of the molecular chaperone Hsc66 from Escherichia coli

Hsc66 from Escherichia coli is a constitutively expressed hsp70 class molecular chaperone whose activity is coupled to ATP binding and hydrolysis. To better understand the mechanism and regulation of Hsc66, we investigated the kinetics of ATP hydrolysis and the interactions of Hsc66 with nucleotides. Steady-state experiments revealed that Hsc66 has a low affinity for ATP (K(m)(ATP) = 12.7 microM) compared with other hsp70 chaperones. The kinetics of nucleotide binding were determined by analyzing changes in the Hsc66 absorbance spectrum using stopped-flow methods at 23 degrees C. ATP binding results in a rapid, biphasic increase of Hsc66 absorbance at 280 nm; this is interpreted as arising from a two-step process in which ATP binding (k(a)(ATP) = 4.2 x 10(4) M(-1) s(-1), k(d)(ATP) = 1.1 s(-1)) is followed by a slow conformational change (k(conf) = 0. 1 s(-1)). Under single turnover conditions, the ATP-induced transition decays exponentially with a rate (k(decay) = 0.0013 s(-1)) similar to that observed in both steady-state and single turnover ATP hydrolysis experiments (k(hyd) = 0.0014 s(-1)). ADP binding to Hsc66 results in a monophasic transition in the absence (k(a)(ADP) = 7 x 10(5) M(-1) s(-1), k(d)(ADP) = 60 s(-1)) and presence of physiological levels of inorganic phosphate (k(a)(ADP(P(i)) = 0.28 x 10(5) M(-1) s(-1), k(d)(ADP(P(i)) = 9.1 s(-1)). These results indicate that ATP hydrolysis is the rate-limiting step under steady-state conditions and is >10(3)-fold slower than the rate of ADP/ATP exchange. Thus, in contrast to DnaK and eukaryotic forms of hsp70 that have been characterized to date, the R if T equilibrium balance for Hsc66 is shifted in favor of the low peptide affinity T state, and regulation of the reaction cycle is expected to occur at the ATP hydrolysis step rather than at nucleotide exchange.     

Kinetic studies of the reduction of yeast glutathione reductase by reduced nicotinamide hypoxanthine dinucleotide phosphate

The reduction of yeast glutathione reductase by reduced nicotinamide hypoxanthine dinucleotide phosphate (NHxDPH) has been examined by stopped-flow kinetic methods. Like reduced glutathione or NADPH, this pyridine nucleotide generates the catalytically active two-electron reduced form of the enzyme. This reductive half-reaction with NHxDPH has only one detectable kinetic step which shows saturation kinetics (Kd = 76 microM), and has a limiting rate constant of 56 s-1. Comparison of stopped-flow and steady-state turnover data indicates that the reductive half-reaction is rate-limiting in the overall catalytic reaction. No evidence was found for a preequilibrium charge-transfer complex between NHxDPH and the active site FAD, like that seen when NADPH is the electron donor.     

A stopped flow transient kinetic analysis of substrate binding and catalysis in Escherichia coli D-3-phosphoglycerate dehydrogenase

Pre-steady state, stopped flow analysis of Escherichia coli D-3-phosphoglycerate dehydrogenase was performed by following the fluorescence of protein tryptophan and the fluorescence resonance energy transfer from protein tryptophan to bound NADH. The results indicate that binding of substrates is ordered, with coenzyme, NADH, binding first. Furthermore, the analysis indicated that there are two sets of sites on the tetrameric enzyme that can be differentiated by their kinetic behavior. NADH binding was consistent with an initial binding event followed by a slow conformational change for each site. The slow conformational change is responsible for the apparent tight binding of NADH to the apoenzyme but is too slow to participate in the catalytic cycle when the enzyme is rapidly turning over. Subsequent binding of the substrate, alpha-ketoglutarate, was characterized by a rapid equilibrium binding event followed by a conformational change for each site. Catalysis in the direction of NAD(+) reduction showed a distinct burst of activity followed by a slow rate of turnover, indicating that the rate-limiting step is after hydride transfer. Catalysis in the direction of NADH oxidation did not display burst kinetics, indicating that the rate-limiting step is at or before the hydride transfer step. The burst data indicated that the rate of NAD(+) reduction (3.8 s(-1)) is similar to the k(cat) of the enzyme (2-3 s(-1)) in that direction. However, analysis of the reaction with deuterated NADH failed to show an effect on the velocity of the reaction with a V(H)/V(D)=1.07+/-0.06. None of the other rates determined by stopped flow analysis could account for the k(cat) of the enzyme in either direction (forward k(cat)=0.01 s(-1), reverse k(cat)=2-3 s(-1)), suggesting that the rate-limiting step in both directions is a conformational change in the enzyme that is not detected optically.     

Branched activation- and catalysis-specific pathways for electron relay to the manganese/iron cofactor in ribonucleotide reductase from Chlamydia trachomatis

A conventional class I (subclass a or b) ribonucleotide reductase (RNR) employs a tyrosyl radical (Y (*)) in its R2 subunit for reversible generation of a 3'-hydrogen-abstracting cysteine radical in its R1 subunit by proton-coupled electron transfer (PCET) through a network of aromatic amino acids spanning the two subunits. The class Ic RNR from the human pathogen Chlamydia trachomatis ( Ct) uses a Mn (IV)/Fe (III) cofactor (specifically, the Mn (IV) ion) in place of the Y (*) for radical initiation. Ct R2 is activated when its Mn (II)/Fe (II) form reacts with O 2 to generate a Mn (IV)/Fe (IV) intermediate, which decays by reduction of the Fe (IV) site to the active Mn (IV)/Fe (III) state. Here we show that the reduction step in this sequence is mediated by residue Y222. Substitution of Y222 with F retards the intrinsic decay of the Mn (IV)/Fe (IV) intermediate by approximately 10-fold and diminishes the ability of ascorbate to accelerate the decay by approximately 65-fold but has no detectable effect on the catalytic activity of the Mn (IV)/Fe (III)-R2 product. By contrast, substitution of Y338, the cognate of the subunit interfacial R2 residue in the R1 <--> R2 PCET pathway of the conventional class I RNRs [Y356 in Escherichia coli ( Ec) R2], has almost no effect on decay of the Mn (IV)/Fe (IV) intermediate but abolishes catalytic activity. Substitution of W51, the Ct R2 cognate of the cofactor-proximal R1 <--> R2 PCET pathway residue in the conventional class I RNRs (W48 in Ec R2), both retards reduction of the Mn (IV)/Fe (IV) intermediate and abolishes catalytic activity. These observations imply that Ct R2 has evolved branched pathways for electron relay to the cofactor during activation and catalysis. Other R2s predicted also to employ the Mn/Fe cofactor have Y or W (also competent for electron relay) aligning with Y222 of Ct R2. By contrast, many R2s known or expected to use the conventional Y (*)-based system have redox-inactive L or F residues at this position. Thus, the presence of branched activation- and catalysis-specific electron relay pathways may be functionally important uniquely in the Mn/Fe-dependent class Ic R2s.     

Inhibition of 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) activity of the antimutagenic human MTH1 protein by nucleoside 5'-diphosphates

The hMTH1 protein, a human homologue of E. coli MutT protein, is an enzyme converting 8-oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) to 8-oxo-2'-deoxyguanosine 5'-monophosphate (8-oxo-dGMP) and inorganic pyrophosphate. It is thought to play an antimutagenic role by preventing the incorporation of promutagenic 8-oxo-dGTP into DNA. As found in our previous investigations, 8-oxo-2'-deoxyguanosine 5'-diphosphate (8-oxo-dGDP) strongly inhibited 8-oxo-dGTPase activity of MTH1. Following this finding, in the present study we have tested the canonical ribo- and deoxyribonucleoside 5'-diphosphates (NDPs and dNDPs) for possible inhibition of 8-oxo-dGTP hydrolysis by hMTH1 extracted from CCRF-CEM cells (a human leukemia cell line). Among them, the strongest inhibitors appeared to be dGDP (Ki=74 microM), dADP (Ki=147 microM), and GDP (Ki=502 microM). Other dNDPs and NDPs, such as dCDP, dTDP, ADP, CDP, and UDP were much weaker inhibitors, with Ki in the millimolar range. Based on the present results and published data, we estimate that the strongest inhibitors, dGDP and dADP, at physiological concentrations not exceeding 5 microM and GDP at mean concentration of 30 microM, taken together, can decrease the cellular hMTH1 enzymatic activity vs. 8-oxo-dGTP (expected to remain below 500 pM) by up to 15%. The other five NDPs and dNDPs tested cannot markedly affect this activity.     

Cotransport of the heterodimeric small subunit of the Saccharomyces cerevisiae ribonucleotide reductase between the nucleus and the cytoplasm

Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in de novo deoxyribonucleotide biosynthesis and is essential in DNA replication and repair. Cells have evolved complex mechanisms to modulate RNR activity during normal cell cycle progression and in response to genotoxic stress. A recently characterized mode of RNR regulation is DNA damage-induced RNR subunit redistribution. The RNR holoenzyme consists of a large subunit, R1, and a small subunit, R2. The Saccharomyces cerevisiae R2 is an Rnr2:Rnr4 heterodimer. Rnr2 generates a diferric-tyrosyl radical cofactor required for catalysis; Rnr4 facilitates cofactor assembly and stabilizes the resulting holo-heterodimer. Upon DNA damage, Rnr2 and Rnr4 undergo checkpoint-dependent, nucleus-to-cytoplasm redistribution, resulting in colocalization of R1 and R2. Here we present evidence that Rnr2 and Rnr4 are transported between the nucleus and the cytoplasm as one protein complex. Tagging either Rnr2 or Rnr4 with a nuclear export sequence causes cytoplasmic localization of both proteins. Moreover, mutations at the Rnr2:Rnr4 heterodimer interface can affect the localization of both proteins without disrupting the heterodimeric complex. Finally, the relocalization of Rnr4 appears to involve both active export and blockage of nuclear import. Our findings provide new insights into the mechanism of DNA damage-induced RNR subunit redistribution.     

Motexafin gadolinium, a tumor-selective drug targeting thioredoxin reductase and ribonucleotide reductase

Motexafin gadolinium (MGd) is a chemotherapeutic drug that selectively targets tumor cells and mediates redox reactions generating reactive oxygen species. Thioredoxin (Trx), NADPH, and thioredoxin reductase (TrxR) of the cytosol/nucleus or mitochondria are major thiol-dependent reductases with many functions in cell growth, defense against oxidative stress, and apoptosis. Mammalian TrxRs are selenocysteine-containing flavoenzymes; MGd was an NADPH-oxidizing substrate for human or rat TrxR1 with a Km value of 8.65 microM (kcat/Km of 4.86 x 10(4) M(-1) s(-1)). The reaction involved redox cycling of MGd by oxygen producing superoxide and hydrogen peroxide. MGd acted as a non-competitive inhibitor (IC50 of 6 microM) for rat TrxR. In contrast, direct reaction between MGd and reduced human Trx was negligible. The corresponding reaction with reduced Escherichia coli Trx was also negligible, but MGd was a better substrate (kcat/Km of 2.23 x 10(5) M(-1) s(-1)) for TrxR from E. coli and a strong inhibitor of Trx-dependent protein disulfide reduction. Ribonucleotide reductase (RNR), a 1:1 complex of the non-identical R1- and R2-subunits, catalyzes the essential de novo synthesis of deoxyribonucleotides for DNA synthesis using electrons from Trx and TrxR. MGd inhibited recombinant mouse RNR activity with either 3 microM reduced human Trx (IC50 2 microM) or 4 mM dithiothreitol (IC50 6 microM) as electron donors. Our results demonstrate MGd-induced enzymatic generation of reactive oxygen species by TrxR plus a powerful inhibition of RNR. This may explain the effects of the drug on cancer cells, which often overproduce TrxR and have induced RNR for replication and repair.     

Histidine 90 function in 4-chlorobenzoyl-coenzyme a dehalogenase catalysis.

4-chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase catalyzes the hydrolytic dehalogenation of 4-CBA-CoA by attack of Asp145 on the C4 of the substrate benzoyl ring to form a Meisenheimer intermediate (EMc), followed by expulsion of chloride ion to form an arylated enzyme intermediate (EAr) and, finally, ester hydrolysis in EAr to form 4-hydroxybenzoyl-CoA (4-HBA-CoA). This study examines the contribution of the active site His90 to catalysis of this reaction pathway. The His90 residue was replaced with glutamine by site-directed mutagenesis. X-ray crystallographic analysis of H90Q dehalogenase complexed with 4-HBA-CoA revealed that the positions of the catalytic groups are unchanged from those observed in the structure of the 4-HBA-CoA-wild-type dehalogenase complex. The one exception is the Gln90 side chain, which is rotated away from the position of the His90 side chain. The vacated His90 site is occupied by two water molecules. Kinetic techniques were used to evaluate ligand binding and catalytic turnover rates in the wild-type and H90Q mutant dehalogenases. The rate constants for 4-CBA-CoA (both 7 microM(-1) x s(-1)) and 4-HBA-CoA (33 and 11 microM(-1) x s(-1)) binding to the two dehalogenases are similar in value. For wild-type dehalogenase, the rate constant for a single turnover is 2.3 s(-1) while that for multiple turnovers is 0.7 s(-1). For H90Q dehalogenase, these rate constants are 1.6 x 10(-2) and 2 x 10(-4) s(-1). The rate constants for EMc formation in wild-type and mutant dehalogenase are approximately 200 s(-1) while the rate constants for EAr formation are 40 and 0.3 s(-1), respectively. The rate constant for hydrolysis of EAr in wild-type dehalogenase is 20 s(-1) and in the H90Q mutant, 0.13 s(-1). The 133-fold reduction in the rate of EAr formation in the mutant may be the result of active site hydration, while the 154-fold reduction in the rate EAr hydrolysis may be the result of lost general base catalysis. Substitution of the His90 with Gln also introduces a rate-limiting step which follows catalysis, and may involve renewing the catalytic site through a slow conformational change.     

An ATP-linked structural change in protein kinase A precedes phosphoryl transfer under physiological magnesium concentrations

The kinetic mechanism for the catalytic subunit of protein kinase A was evaluated using physiological concentrations of free magnesium (0.5 mM) and a rapid quench flow technique. When the enzyme is pre-equilibrated with ATP, the peptide substrate, LRRASLG (Kemptide), is phosphorylated in a biphasic manner with a rapid, exponential "burst" phase (kb) followed by a slower, linear phase (kL) that corresponds to the steady-state kinetic rate. Both the amplitude and the substrate-rate dependence of the initial, burst phase indicate that the rate of phosphoryl transfer is fast (approximately 500 s-1) and does not limit turnover (45 s-1). Viscosity studies indicate that, while Kemptide is in rapid equilibrium, ATP does not exchange rapidly with the active site and kcat/KATP is limited by the rate constant for nucleotide encounter. When the pre-steady-state kinetic experiments are initiated with ATP, a lag phase is observed at low ATP concentrations consistent with rate-limiting association. At high ATP concentrations (>1 mM), a burst phase is observed but the rate and amplitude are low on the basis of the bimolecular rate constant for ATP association and the rate constant for phosphoryl transfer. The kinetic data indicate that the phosphoryl transfer step is fast at physiological magnesium concentrations, but an ATP-linked conformational change precedes this step, limiting the burst phase rate constant. Simulations of the pre-steady-state kinetic transients indicate that turnover (45 s-1) is limited both by net product release (70 s-1) and by this structural change (170 s-1). This structural change may also occur at high free magnesium concentrations, but it must be significantly faster than 170 s-1 and, consequently, not rate-limiting for turnover (kcat = 20 s-1 at 10 mM free Mg2+). We propose that this conformational event is an obligatory component of the kinetic pathway and includes a movement of the catalytic residues necessary for supporting phosphoryl group donation.