Rate-limiting Step Of Reaction Research Articles

Limited internal friction in the rate-limiting step of a two-state protein folding reaction.

Small, single-domain proteins typically fold via a compact transition-state ensemble in a process well fitted by a simple, two-state model. To characterize the rate-limiting conformational changes that underlie two-state folding, we have investigated experimentally the effects of changing solvent viscosity on the refolding of the IgG binding domain of protein L. In conjunction with numerical simulations, our results indicate that the rate-limiting conformational changes of the folding of this domain are strongly coupled to solvent viscosity and lack any significant "internal friction" arising from intrachain collisions. When compared with the previously determined solvent viscosity dependencies of other, more restricted conformational changes, our results suggest that the rate-limiting folding transition involves conformational fluctuations that displace considerable amounts of solvent. Reconciling evidence that the folding transition state ensemble is comprised of highly collapsed species with these and similar, previously reported results should provide a significant constraint for theoretical models of the folding process.

The reaction mechanism for CD38. A single intermediate is responsible for cyclization, hydrolysis, and base-exchange chemistries.

Human recombinant CD38 catalyzes the formation of both cyclic ADP-ribose and ADP-ribose products from NAD+ and hydrolyzes cyclic ADP-ribose to ADP-ribose. The corresponding GDP products are formed from NGD+. The enzyme was characterized by substrate and inhibition kinetics, exchange studies, rapid-quench reactions, and stopped-flow-fluorescence spectroscopy to establish the reaction mechanism and energetics for individual steps. Noncyclizable substrates NMN+ and nicotinamide-7-deaza-hypoxanthine dinucleotide (7-deaza NHD+) were rapidly hydrolyzed by the enzyme. The kcat for NMN+ was 5-fold higher than that of NAD+ and has the greatest reported kcat of any substrate for CD38. 7-deaza-NHD+ was hydrolyzed at approximately one-third the rate of NHD+ but does not form a cyclic product. These results establish that a cyclic intermediate is not required for substrate hydrolysis. The ratio of methanolysis to hydrolysis for cADPR and NAD+ catalyzed by CD38 increases linearly with MeOH concentration. Both reactions produce predominantly the beta-methoxy riboside compound, with a relative nucleophilicity of MeOH to H2O of 11. These results indicate the existence of a stabilized cationic intermediate for all observed chemistries in the active site of CD38. The partitioning of this intermediate between cyclization, hydrolysis, and nicotinamide-exchange unites the mechanisms of CD38 chemistries. Steady-state and pre-steady-state parameters for the partition and exchange mechanisms allowed full characterization of the reaction coordinate. Stopped-flow methods indicate a burst of cGDPR formation followed by the steady-state reaction rate. A lag phase, which was NGD+ concentration dependent, was also observed. The burst size indicates that the dimeric enzyme has a single catalytic site formed by two subunits. Pre-steady-state quench experiments did not detect covalent intermediates. Nicotinamide hydrolysis of NGD+ precedes cyclization and the chemical quench decomposes the enzyme-bound species to a mixture of cyclic and hydrolysis products. The time dependence of this ratio indicated that nicotinamide bond-breakage occurs 4 times faster than the conversion of the intermediate to products. Product release is the overall rate-limiting step for enzyme reaction with NGD+.

Ab Initio Molecular Orbital Study of the Unified Mechanism and Pathways for Gas−Carbon Reactions

Ab Initio molecular orbital calculations are performed at the B3LYP/6-31G(d)/HF/3-21G(d) calculation level on graphite and oxygen intermediates on and near the edges of graphite. The results are in agreement with experimental data for structural geometry parameters, vibrational frequencies, and, more importantly, bond energies. Three intermediates are studied: semiquinone, carbonyl, and an off-plane, epoxy oxygen. The rate-limiting step for all gasification reactions by oxygen-containing gases is the breakage of C−C bonds to free CO from the semiquinone intermediate. The energy for this C−C bond is near 80 kcal/mol, which is close to the experimental activation energy for the reactions with CO2 and H2O. The C−C bond is weakened by 33% by the adjacent epoxy oxygen, to a bond energy of nearly 53 kcal/mol. This extent of weakening in the C−C bond energy coincides with the decrease in the experimental activation energy from 85 kcal/mol for the reactions with CO2 and H2O to 58 kcal/mol for the reaction with O...

A Molecular Modeling Study of the Catalytic Mechanism of Haloalkane Dehalogenase. 2. Quantum Chemical Study of Complete Reaction Mechanism

Haloalkane dehalogenase is a bacterial enzyme, which catalyzes hydrolytic cleavage of the carbon−halogen bond of haloalkanes. Quantum mechanical calculations at the semiempirical level using the MOPAC/DRIVER methodology were applied to study the enzymatic hydrolysis of 1,2-dichloroethane to 2-chloroethanol. In our previous study, the first SN2 step of dehalogenation reaction was investigated (Damborský, J.; Kutý, M.; Němec, M.; Koča, J. A Molecular Modeling Study of the Catalytic Mechanism of Haloalkane Dehalogenase: 1. Quantum Chemical Study of the First Reaction Step. J. Chem. Inf. Comput. Sci. 1997, 37, 562−568). The present contribution explores the complete three-step reaction to determine the rate-limiting reaction step and to investigate the importance of active site residues for the kinetics and thermodynamics of the hydrolysis. The nucleophilic addition (AdN) step has the highest energy barrier, which is in qualitative agreement with experimental rates, assigning the second hydrolytic step as the rate-limiting one. In order to establish the catalytically important active-site residues, Mulliken charges of selected active-site atoms were monitored along the reaction pathway. A significant change in charges on the hydrogen atoms of Trp125, Trp175, and Phe172 active-site residues was observed. These residues interact with the halide ion released during the SN2 step. Changes in charges on the hydrogen atoms of Trp125 and Glu56 prove the significance of those residues in the stabilization of the partial charge developed on the oxygen atom of the nucleophilic aspartate (Asp124). The same methodology confirmed the importance of the charge relay system (Asp124, His289, and Asp260 residues) in the base-hydrolysis reaction (AdN step).

Kinetic isotope effects as probes of the mechanism of galactose oxidase.

Galactose oxidase (GO) is a member of the family of radical-coupled copper oxidases, enzymes containing a free radical coordinated to copper in the active site. In catalysis GO cycles between an oxidized state (comprising Cu(II) with a unique cysteinyl-tyrosine radical) and a reduced state (comprising Cu(I) with the singlet cysteinyl-tyrosine) as it catalyzes the two-electron oxidation of alcohols to aldehydes and the subsequent reduction of O2 to H2O2. A ping-pong mechanism involving radical intermediates has been proposed for GO catalysis. Previous steady-state kinetics studies have demonstrated a KIE of 7-8 that was attributed to substrate oxidation, a process involving the stereospecific abstraction of the pro-S hydrogen from the 6-hydroxymethyl group of galactose. We have used rapid kinetics methods to measure the anaerobic reduction of GO substrate at 4 degreesC and carry out enzyme-monitored turnover experiments using 6-protio and 6-deutero substrates, both in H2O and D2O. At concentrations below Km, the apparent second-order rate constant for protio-substrate oxidation, kred, was 1.59 x 10(4) M-1 s-1, while that for deuterated substrate was 7.50 x 10(2) M-1 s-1, a KIE of 21.2. Steady-state measurements of oxygen consumption at low galactose concentrations reveal an unusually large isotope effect (kH/kD = 22.5 +/- 2) for oxidation of 1-O-methyl-6, 6'-di-[2H]-alpha-d-galactopyranoside, and at high galactose concentrations, where the oxygen half-reaction is rate-limiting in catalysis, a surprisingly large KIE (kH/kD = 8 +/- 1) for the reduction of O2 to H2O2. There is no detectable solvent isotope effect (<5%) on any of these measurements. This shows that there are no exchangeable protons involved in any kinetically significant step and that the hydrogen atom removed from galactose is not lost to solvent during catalysis; instead, it also participates in the rate-limiting step of the subsequent reaction with oxygen. At concentrations below Km, apparent second-order rate constants for protio-substrate oxidation (kred = 1.5 x 10(4) M-1 s-1) and O2 reduction (kox = 8 x 10(6) M-1 s-1) have been estimated from measurements both by steady-state oxygen electrode and by enzyme-monitored turnover. This is completely consistent with the anaerobic studies mentioned above. Our results show that the enzyme is essentially fully oxidized while in steady-state turnover, consistent with the reduction step being nearly fully rate-limiting at practical substrate concentrations, due to the very fast reaction with physiological concentrations of O2. Overall, the catalytic reaction is in concordance with a ping-pong mechanism. The large KIE associated with reduction of the enzyme in all three methods appears to reflect hydrogen atom radical abstraction by the active site tyrosine radical in the rate-determining step, in agreement with the previously proposed radical mechanism for GO. The KIE determined at low substrate concentrations (where oxidation of substrate is rate determining) from steady-state oxygen consumption measurements, varies from 22.5 at 4 degreesC to 13 at 45 degreesC, consistent with tunneling being involved in the hydrogen atom transfer step.

Substrate specificity of sheep liver sorbitol dehydrogenase.

The substrate specificity of sheep liver sorbitol dehydrogenase has been studied by steady-state kinetics over the range pH 7-10. Sorbitol dehydrogenase stereo-selectively catalyses the reversible NAD-linked oxidation of various polyols and other secondary alcohols into their corresponding ketones. The kinetic constants are given for various novel polyol substrates, including L-glucitol, L-mannitol, L-altritol, D-altritol, D-iditol and eight heptitols, as well as for many aliphatic and aromatic alcohols. The maximum velocities (kcat) and the substrate specificity-constants (kcat/Km) are positively correlated with increasing pH. The enzyme-catalysed reactions occur by a compulsory ordered kinetic mechanism with the coenzyme as the first, or leading, substrate. With many substrates, the rate-limiting step for the overall reaction is the enzyme-NADH product dissociation. However, with several substrates there is a transition to a mechanism with partial rate-limitation at the ternary complex level, especially at low pH. The kinetic data enable the elucidation of new empirical rules for the substrate specificity of sorbitol dehydrogenase. The specificity-constants for polyol oxidation vary as a function of substrate configuration with D-xylo> D-ribo > L-xylo > D-lyxo approximately L-arabino > D-arabino > L-lyxo. Catalytic activity with a polyol or an aromatic substrate and various 1-deoxy derivatives thereof varies with -CH2OH > -CH2NH2 > -CH2OCH3 approximately -CH3. The presence of a hydroxyl group at each of the remaining chiral centres of a polyol, apart from the reactive C2, is also nonessential for productive ternary complex formation and catalysis. A predominantly nonpolar enzymic epitope appears to constitute an important structural determinant for the substrate specificity of sorbitol dehydrogenase. The existence of two distinct substrate binding regions in the enzyme active site, along with that of the catalytic zinc, is suggested to account for the lack of stereospecificity at C2 in some polyols.

Effects of 3′-phosphoadenosine 5′-phosphate on the activity and folding of phenol sulfotransferase

Known spectroscopic and kinetic data are used to formulate pathways of the physiological and transfer reactions and the substrate inhibition of phenol sulfotransferase. Kinetic mechanisms indicate that release of PAP from enzyme complex is required for the physiological reaction but not for the transfer reaction. The pathways explain rate difference between the physiological and transfer reactions since the release of PAP is the rate-limiting step of the former reaction. Two enzyme species of phenol sulfotransferase which distinguish the physiological and transfer reaction were found to involve the binding of PAP. Differences between two forms of phenol sulfotransferase, α and β, indicate that they assemble through different folding process. It is demonstrated that only α enzyme renatures in the presence of PAP and β enzyme renatures only in the absence of PAP in vitro. In the over-expressed system, formation of α and β phenol sulfotransferase is also dependent on the availability of PAP in Escherichia coli. It is concluded that folding of phenol sulfotransferase is assisted by PAP to form α enzyme. In the absence of PAP, β form of phenol sulfotransferase is produced.

Kinetics of methylation and binding of DNA by the EcoRV adenine-N6 methyltransferase

The EcoRV DNA methyltransferase (M.EcoRV) specifically methylates the first adenine within its recognition sequence GATATC. Methylation rates of DNA by this enzyme are strongly influenced by the length of oligonucleotide substrates employed. If substrates > 20 bp compared to a 12mer substrate, the kcat/Kmincreases 100-fold, although the enzyme does not contact more than 12 base-pairs on the DNA. Single-turnover rates are higher than kcatvalues. M.EcoRV binding to DNA is fast but dissociation from the DNA is slow, demonstrating that the multiple-turnover rate is limited by the rate of product release. The kinetics of DNA binding by M.EcoRV are not in accordance with the thermodynamics binding constant, suggesting that the M.EcoRV-DNA complex is involved in a slow conformational change. The salt dependence of DNA binding is different for non-specific substrates (d ln(KAss)/d ln(cNaCl) = −2, indicative of electrostatic interactions) and specific substrates (d ln(KAss)/d ln(cNaCl) = +1, indicative of hydrophobic interactions). This result demonstrates that the M.EcoRV-DNA complex has a different conformation in both binding modes. M.EcoRV does not discriminate between hemimethylated and unmethylated substrates. Using the 20mer we have analyzed the temperature and pH dependence of the single-turnover rate constant of M.EcoRV-DNA methylation by M.EcoRV has an activation energy of 40 kJ/mol and its rate increases with increasing pH. The pH dependence reveals the presence of an ionizable residue with a pKaof 7.9, which must be unprotonated for catalysis. The rates of DNA methylation remain unchanged if an abasic site is introduced instead of the thymidine residue that is base-paired to the target adenine, demonstrating that flipping out the target adenine cannot contribute to the rate-limiting step of the enzymatic reaction.

Reactive uptake of HNO 3 and H 2SO 4 in sea-salt (NaCl) particles

Kinetic information on the substitution of sea-salt chloride by nitrate was deduced from a smog chamber investigation on the reaction of airborne NaCl with HNO 3. It was found that a measurable reaction only occurred when the NaCl-particles were present in the form of droplets. The substitution of chloride by nitrate was independent of size, which shows that the generation of the product (HCI gas) was the rate-limiting reaction step. The rate of this reaction was more than an order of magnitude slower than the rate at which nitric acid can reach the droplets. The substitution of chloride by sulphate, in a reaction between H 2SO 4 and NaCl, depended on particle size from which it was concluded that the transport of H 2SO 4 to the aerosol was the rate-limiting process. The difference in reaction of the two acids is explained by the fact that sulphuric acid is a condensable species, whereas nitric acid is a gas. From the amount of sulphate as a function of size an uptake coefficient for the condensing sulphuric acid was deduced as 0.1 or higher.

Kinetics and mechanism of chromic acid oxidation of oxalic acid in absence and presence of different acid media. A kinetic study

Kinetics and mechanism of the reaction of Cr(VI) with oxalic acid have been studied in presence and absence of H2SO4, HClO4, and CH3COOH by monitoring the formation of Cr(III)-oxalic acid complex at 560 nm. The effect of total [oxalic acid], [Cr(VI)], [H2SO4], [HClO4], and [CH3COOH] on the reaction rate was determined at 30°C. Formation of carbon dioxide was also confirmed. The oxidation rate increases with [oxalic acid] and [CH3COOH] while it decreases with [H2SO4], [HClO4], and pH. The rate law governing the oxidation of oxalic acid over a wide range of conditions is rate=k1 Kes1 [oxalic acid]T [Cr(VI)]T 1+Kes1 [oxalic acid]T, where only undissociated oxalic acid is kinetically active. Kinetic evidence for the formation of a Cr(VI)(SINGLEBOND)oxalic acid 1:1 complex has been obtained and the equilibrium constant for their formation has been determined. The 1:1 complex exists most likely in an open chain form. The rate-limiting step of the oxidation reaction involves the breaking of the C(SINGLEBOND)C bond in the 1:2 complex. Oxidizing ability of Cr(VI) species have been discussed. Mechanism with the associated reaction kinetics is assigned. © 1998 John Wiley & Sons, Inc. Int J Chem Kinet 30: 335–340, 1998

Kinetics and mechanism of chromic acid oxidation of oxalic acid in absence and presence of different acid media. A kinetic study

Kinetics and mechanism of the reaction of Cr(VI) with oxalic acid have been studied in presence and absence of H2SO4, HClO4, and CH3COOH by monitoring the formation of Cr(III)-oxalic acid complex at 560 nm. The effect of total [oxalic acid], [Cr(VI)], [H2SO4], [HClO4], and [CH3COOH] on the reaction rate was determined at 30°C. Formation of carbon dioxide was also confirmed. The oxidation rate increases with [oxalic acid] and [CH3COOH] while it decreases with [H2SO4], [HClO4], and pH. The rate law governing the oxidation of oxalic acid over a wide range of conditions is rate=k1Kes1 [oxalic acid]T [Cr(VI)]T 1+Kes1 [oxalic acid]T, where only undissociated oxalic acid is kinetically active. Kinetic evidence for the formation of a Cr(VI)(SINGLEBOND)oxalic acid 1:1 complex has been obtained and the equilibrium constant for their formation has been determined. The 1:1 complex exists most likely in an open chain form. The rate-limiting step of the oxidation reaction involves the breaking of the C(SINGLEBOND)C bond in the 1:2 complex. Oxidizing ability of Cr(VI) species have been discussed. Mechanism with the associated reaction kinetics is assigned. © 1998 John Wiley & Sons, Inc. Int J Chem Kinet 30: 335–340, 1998

The interaction of mitochondria with pulses of calcium.

Mitochondria seem to be distributed within cells so as to provide the necessary amounts of ATP when and where it is needed. In muscle tissue many mitochondria are located near the contracting filaments. In cells which make and secrete large amounts of protein many mitochondria are found near the rough endoplasmic reticulum. In neurons mitochondria use energy to move in an anterograde direction through the axon on microtubules and later return in the retrograde direction, distributing themselves, in the process, along the axon. In all cells there are a significant number of mitochondria near the nucleus. For years, we have known of the mitochondrial calcium uniporter, of the Na+-dependent and Na+independent efflux mechanisms, and of the permeability transition pore [2]. More recently, work first with aequorin and then with artificially synthesized fluorescent calcium indicators showed that the levels of calcium within the cytosol of cells are low – in the range of 100 nM, except during calcium transients or pulses that often pass like waves through the cytosol [1]. The calcium within these pulses or transients comes, in large part, from release from intracellular stores, replenished by calcium influx from the extracellular space through calcium channels. These intracellular stores are found in the sarcoplasmic reticulum of muscle tissue and generally within the endoplasmic reticulum of other tissue. During these calcium pulses or transients the cytosolic calcium concentration ([Ca2+]) rises into the range from 0.5 to over 1.0 μM. In restricted regions near the pores effluxing calcium from intracellular stores, the [Ca2+] can go even higher. Several reactions within the intramitochondrial metabolic pathways have been found to be activated by calcium[1]. Of these, those catalyzed by three dehydrogenases coupled to the TCA cycle (pyruvate dehydrogenase, alpha keto gluterate dehydrogenase, and isocitrate dehydrogenase) are the best known; however, the electron transport system within liver mitochondria, the F1 ATPase, and the adenine nucleotide translocase have also been found to be stimulated by calcium. The advocates of metabolic control theory support the view that there is not a single rate-limiting step for the intramitochondrial metabolic reactions and that calcium acts to coordinate the activation of several steps among which rate limitation is shared so as to increase the rate of ATP production (for review, see [1]). The kinetics of the influx and efflux of calcium through the uniporter and the Na+-dependent and Na+independent efflux mechanisms are known for the most frequently studied types of mitochondria (liver and heart) [2]. Calculations of the amount of calcium taken up by mitochondria from calcium pulses like those observed in cells originally led to the conclusion that this calcium uptake would be insufficient to activate the intramitochondrial calcium-dependent metabolic reactions, except in those cases where the mitochondria are located near the intracellular stores from which the calcium in the calcium pulses is

A functional role for histidyl residues of the UDP-glucuronic acid carrier in rat liver endoplasmic reticulum membranes.

Previous studies have documented the presence of protein-mediated transport of UDP-glucuronic acid (UDP-GlcUA) in rat liver endoplasmic reticulum (ER). To determine the crucial amino acids of the membrane transporter and evaluate their function in regulating the glucuronidation reaction, we examined the effect of histidyl-specific irreversible inhibitors on the uptake of radiolabeled UDP-GlcUA in rat liver ER. Inactivation of uptake (initial rate) was more pronounced with hydrophobic reagents [diethyl pyrocarbonate (DEPC), p-bromophenacyl bromide] as compared to the more hydrophilic reagent (p-nitrobenzenesulfonic acid methyl ester). DEPC was used to further characterize the inhibition because of its greater specificity for protein histidyl residues. While initial [14C]UDP-GlcUA uptake rates were diminished by DEPC treatment of intact microsomes, the accumulation of isotope at equilibrium was not significantly affected, indicating no loss of vesicle integrity. A pKa of approximately 7 for the modified residue(s) of the transporter supported the alkylation of imidazole moieties. Protection against inactivation was observed with UDP-GlcUA as well as other nucleotide-sugars known for their interaction with this transporter. Uptake activity of the transporter (Vmax) but not UDP-GlcUA binding (Km) was affected by a limited inactivation. Furthermore, a partial inactivation of the transporter impaired the binding of the photoaffinity label [beta-32P]5-azido-UDP-GlcUA to UDP-glucuronosyltransferases (UGTs) in intact, but not in detergent-disrupted, ER vesicles. These results demonstrate the involvement of histidyl residue(s) in the UDP-GlcUA uptake process in rat liver ER, provide additional evidence for the lumenal orientation of the UGT active site, and support the view that translocation of the UGT cosubstrate is a rate-limiting step of the glucuronidation reaction.

Characterization of cholesterol oxidase from Streptomyces hygroscopicus and Brevibacterium sterolicum.

The FAD-containing enzyme cholesterol oxidase catalyzes the oxidation and isomerization of 3beta-hydroxysteroids having a trans double bond at delta5-delta6 of the steroid ring backbone to the corresponding delta4-3-ketosteroid. Two representative enzymes of this family, namely cholesterol oxidase from Streptomyces hygroscopicus (SCO) and the recombinant enzyme from Brevibacterium sterolicum (BCO) expressed in Escherichia coli, have been characterized herein in their chemical, physical, and biochemical properties. In the native form, both enzymes are monomeric (55 kDa), acidic (pI 4.4-5.1) and contain oxidized FAD (peaks in the 370-390-nm and 440-470-nm regions). Marked differences exist between the oxidized, reduced, and (red) anion semiquinone spectra of the two enzymes, suggesting substantial differences in the flavin microenvironment. Both enzymes form reversibly flavin N(5)-sulfite adducts via measurable k(on) and k(off) steps. BCO has a higher affinity for sulfite (Kd approximately 0.14 mM) compared to SCO (approximately 24 mM). This correlates well with the midpoint redox potentials of the bound flavin, which in the case of BCO are about 100 mV more positive than for SCO. Both enzymes show a high pKa (approximately 11.0) for the N(3) position of FAD. With both enzymes, the rearrangement of 5-cholesten-3-one to 4-cholesten-3-one is not rate limiting indicating that the rate-limiting step of the overall reaction is not the isomerization. The absence of the double bond in the steroid molecule does not significantly affect turnover and affinity for the substrate, whereas both these parameters are affected by a decreasing length of the substrate C17 chain.

Catalytic Mechanism and Role of Hydroxyl Residues in the Active Site of Theta Class Glutathione S-Transferases: INVESTIGATION OF SER-9 AND TYR-113 IN A GLUTATHIONES-TRANSFERASE FROM THE AUSTRALIAN SHEEP BLOWFLY,LUCILIA CUPRINA

Spectroscopic and kinetic studies have been performed on the Australian sheep blowfly Lucilia cuprina glutathione S-transferase (Lucilia GST; EC 2.5.1.18) to clarify its catalytic mechanism. Steady state kinetics of Lucilia GST are non-Michaelian, but the quite hyperbolic isothermic binding of GSH suggests that a steady state random sequential Bi Bi mechanism is consistent with the anomalous kinetics observed. The rate-limiting step of the reaction is a viscosity-dependent physical event, and stopped-flow experiments indicate that product release is rate-limiting. Spectroscopic and kinetic data demonstrate that Lucilia GST is able to lower the pKa of the bound GSH from 9.0 to about 6.5. Based on crystallographic suggestions, the role of two hydroxyl residues, Ser-9 and Tyr-113, has been investigated. Removal of the hydroxyl group of Ser-9 by site-directed mutagenesis raises the pKa of bound GSH to about 7.6, and a very low turnover number (about 0.5% of that of wild type) is observed. This inactivation may be explained by a strong contribution of the Ser-9 hydroxyl group to the productive binding of GSH and by an involvement in the stabilization of the ionized GSH. This serine residue is highly conserved in the Theta class GSTs, so the present findings may be applicable to all of the family members. Tyr-113 appears not to be essential for the GSH activation. Stopped-flow data indicate that removal of the hydroxyl group of Tyr-113 does not change the rate-limiting step of reaction but causes an increase of the rate constants of both the formation and release of the GSH conjugate. Tyr-113 resides on alpha-helix 4, and its hydroxyl group hydrogen bonds directly to the hydroxyl of Tyr-105. This would reduce the flexibility of a protein region that contributes to the electrophilic substrate binding site; segmental motion of alpha-helix 4 possibly modulates different aspects of the catalytic mechanism of the Lucilia GST.

Water Catalyzed Hydrolysis ofp-Nitrotrifluoroacetanilide and Trifluoroacetanilide. Carbonyl18O-Exchange Does Not Accompany the Water Reaction

The water catalyzed hydrolyses of p-nitrotrifluoroacetanilide (3) and trifluoroacetanilide (8) were studied at various pH's and temperatures. The activation parameters for the water reactions of 3 and 8 are: ΔH⧧ = 14.4 ± 0.6 and 11.7 ± 0.3 kcal/mol and ΔS⧧ = −36.1 ± 1.8 and −52.3 ± 0.7 cal/mol·K, respectively. These are consistent with reactions that involve considerable restriction of degrees of freedom of the solvent/substrate in the transition state. A proton inventory analysis of the rate constants for hydrolysis of 3 in media of different mole fraction D2O indicates the process involves two or more protons in flight or undergoing loosening of their bonding in the transition state. 18O-Labeled amides were subjected to the hydrolytic conditions for various times up to 3 half-times of hydrolysis and recovered. Mass analysis showed that the 18O content in the recovered amide did not change during the course of the reaction. All the data support a process where the rate-limiting step for the water reaction involves a concerted or nearly concerted formation of a diol which undergoes subsequent C−N cleavage in preference to OH expulsion.

Role of glutamine-151 of human immunodeficiency virus type-1 reverse transcriptase in RNA-directed DNA synthesis.

Glutamine-151 of HIV-1 RT has been shown to be a catalytically important residue through the characterization of its mutant phenotype Glu151Ala (Sarafianos et al., 1995a). To further understand the role of this residue, we have extended this analysis to include polymerization on natural RNA template in addition to DNA template. We find that Q151A mutant exhibited a severe reduction in the polymerase activity without any significant effect on the affinity for dNTP substrate. Unlike DNA-directed reactions, the rate-limiting step for RNA-directed reactions does not appear to be either at the dNTP binding step or the chemical step. Analysis of the products formed on natural heteromeric HIV-genomic RNA template annealed with an 18-mer DNA primer with a sequence complementary to the primer binding site (PBS) has shown that addition of nucleotides is nonlinear with time since the enzyme appears to stall on the RNA template following the incorporation of the first nucleotide. The Q151A mutant was found to be nearly devoid of pyrophosphorolytic activity on a RNA-PBS template-primer. Similar properties have been previously reported for a mutant of R72 (R72A) of HIV-1 RT (Sarafianos et al., 1995b). However, R72 was implicated in stabilizing the transition state ternary complex before and after the phosphodiester bond formation (Kaushik et al., 1996; Sarafianos et al., 1995b). Our results with Q151A suggest that the side chain of Q151 may help stabilize the side chain of R72, and the loss of pyrophosphorolysis activity observed with the Q151 mutant may be the indirect manifestation of this stabilizing effect on R72. These observations point to the functional interdependence of residues Q151 and R72 in the polymerase function of the enzyme. An analysis of the 3D model structure of HIV-1 RT bound to DNA-DNA and RNA-DNA template-primer reveals that the guanidine hydrogen of R72 seems to stabilize Q151 by hydrogen bonding with its amide oxygen. A systematic conformational search of the side chain of Q151 also suggests a stable orientation where its specific interaction with the base of the RNA template may aid in stabilizing it.

Rescue of a mutant G-protein by substrate-assisted catalysis.

Signaling by guanine-nucleotide-binding proteins (G-proteins) occurs when they are charged with GTP, while hydrolysis of the bound nucleotide turns the signaling off. Despite a wealth of biochemical and structural information, the mechanism of GTP hydrolysis by G-proteins remains controversial. We have employed substrate-assisted catalysis as a novel approach to study catalysis by G-proteins. In these studies, we have used diaminobenzophenone-phosphonoamidate-GTP, a unique GTP analog bearing the functional groups that are missing in the GTPase-deficient [Leu227]G(s alpha) mutant. This mutant, found in various human tumors, fails to hydrolyze GTP for an extended period. In contrast, the GTP analog is hydrolyzed by this mutant and by the wild-type enzyme at the same rate. On the other hand, modification of G(s alpha) by cholera toxin, which catalyses ADP-ribosylation of Arg201 of G(s alpha), decreased the rates of hydrolysis of both GTP and its analog by 95%. These results attest to the specificity of the GTP analog as a unique substrate for the [Leu227]G(s alpha) mutant and to the essential role of Gln227 in GTP hydrolysis. Furthermore, the finding that the GTP analog was hydrolyzed at the same rate as GTP by the wild-type enzyme, favors a model in which formation of a pentavalent transition state intermediate, presumably stabilized by the catalytic glutamine, is not the rate-limiting step of the GTPase reaction.

Escherichia coli Diacylglycerol Kinase Is an Evolutionarily Optimized Membrane Enzyme and Catalyzes Direct Phosphoryl Transfer

In this contribution the kinetic mechanism and substrate specificity of Escherichia coli diacylglycerol kinase were examined. Steady state kinetic studies were carried out under mixed micellar conditions using a novel continuous coupled assay system. The kinetic data were consistent with a random equilibrium mechanism, implying that diacylglycerol kinase catalyzes direct phosphoryl transfer from MgATP to diacylglycerol. This was supported by failure to detect an enzyme-phosphate covalent intermediate and by the observation that the bisubstrate analog adenosine 5'-tetraphosphoryl-3-O-(1,2-dihexanoyl)-sn-glycerol inhibits the enzyme (Ki << Km,DAG). While diacylglycerol kinase's kcat/Km is modest compared with the efficiency of many water-soluble enzymes, the enzyme nevertheless appears to be an evolutionarily optimized biocatalyst in the sense that its chemical reaction rate approaches the substrate diffusion-controlled limit. The in vivo rate-limiting step of DAGK's reaction appears to be, in part, the transbilayer diffusion of diacylglycerol from the outer leaflet to the inner leaflet of the cytoplasmic membrane where DAGK's active site is located. DAGK was observed to maintain a high nucleotide substrate specificity, with most of this specificity being expressed in the form of reductions in kcat for ATP analogs.

Reactions of Nitrate Salts with Ammonia in Supercritical Water

Reactions involving nitrate salts and ammonia were investigated in supercritical water at temperatures from 450 to 530 C and pressures near 300 bar. Reaction products included nitrite, nitrogen gas, and nitrous oxide. Observed reaction rates and product distributions provided evidence for a free-radical reaction mechanism with NO{sub 2}, NO, and NH{sub 2}{center_dot} as the primary reactive species at supercritical conditions. In the proposed elementary mechanism, the rate-limiting reaction step was determined to be the hydrolysis of MNO{sub 3} species, which resulted in the formation of nitric acid and subsequently NO{sub 2}. A simple second-order reaction model was used to represent the data. In developing an empirical kinetic model, nitrate and nitrate were lumped as an NO{sub x}{sup {minus}} reactant. Empirical kinetic parameters were developed for four MNO{sub x}/NH{sub 3} reacting systems, assuming first orders in both NH{sub 3} and NO{sub x}{sup {minus}}. Observed MNO{sub x}/NH{sub 3} reaction rates and mechanisms suggest immediately a practical significance of these reactions for nitrogen control strategies in supercritical water oxidation processes.