Serine activation is the rate limiting step of tRNASer aminoacylation by yeast seryl tRNA synthetase.

Using the quenched flow technique the mechanism of seryl tRNA synthetase action has been investigated with respect to the presteady state kinetics of individual steps. Under conditions where the strong binding sites of the enzyme are nearly saturated and the steady state turnover number is about 1 s-1, rate constants of four different processes have been determined: steps connected with substrate associations are relatively slow (12 s-1 for the entire process); activation of serine is the rate determining step (about 1.2 s-1 in presence of tRNASer); whereas the transfer of serine onto tRNASer (35 s-1) and the dissociation of seryl tRNASer (70 s-1) are fast. Similar kinetic parameter seem to hold also for the steady state reactions. This conclusion is based on a detailed study of the substrate, product, and Mg2+ concentration dependence of the transfer reaction. The results also indicate that a second serine binding site is operative. Since the transfer of serine from a preformed adenylate complex onto tRNASer is fast, seryl adenylate seems to be a kinetically competent intermediate of the aminoacylation reaction although, of course, alternative mechanisms cannot be excluded.     

Editing mechanisms in protein synthesis. Rejection of valine by the isoleucyl-tRNA synthetase.

Although the isoleucyl-tRNA synthetase from Escherichia coli (IRS) does not catalyze the overall mischarging of tRNAIle with valine, it does undergo the first step of the reaction, the formation of an IRS-Val-AMP complex. The addition of tRNAIle to this complex leads to its quantitative hydrolysis and the IRS acts as an ATP pyrophosphate in the presence of valine and tRNAIle (Baldwin, A.N., and Berg, P. (1966), J. Biol. Chem. 241, 839). It is shown that during the ATP pyrophosphatase reaction: (a) IRS forms an IRS-Val-AMP complex; (b) the turnover number of the ATP pyrophosphatase reaction is the same at the rate constant for the transfer of isoleucine from IRS-Ile-AMP to tRNAIle over a wide range of temperature and pH; (c) mischarged Val-tRNAIle is hydrolyzed by IRS with a turnover number of 10 s-1 at pH 7.78 and 25 degrees C, compared with a value of 1.2 s-1 for the transfer of isoleucine from IRS-Ile-AMP to tRNA or for the ATP pyrophosphatase reaction. Although this appears to be consistent with an editing mechanism in which there is a slow transfer of the valine from the IRS-Val-AMP to tRNAIle follwed by the rapid hydrolytic step, as recently found for the rejection of threonine by the valyl-tRNA synthetase, there is an inconsistency. This scheme predicts that on mixing IRS.[14C]Val-AMP with tRNAIle there should be a transient misacylation of the tRNA such that about 10% of the [14C]Val is present as [14C]Val-tRNAIle at the peak. But 0.8% or less is found. This could possibly be caused by the IRS having a higher hydrolytic activity during the mischarging reaction than is measured on mixing the unligated enzyme with performed Val-tRNAIle. Alternatively, a two-stage editing mechanism must be considered in which the majority of the Val-AMP is destroyed before the transfer to tRNA in the major editing step, while the hydrolytic activity of the IRS towards Val-tRNAIle is a second editing step to mop up any mischarged tRNA formed by the Val-AMP escaping the first editing step. It is shown that the "kinetic proofreading" mechanism of Hopfield is not consistent with the experimental data.     

Determination of the mechanism of the argininosuccinate synthetase reaction by static and dynamic quench experiments

The reactions catalyzed by argininosuccinate synthetase have been examined by the use of static and dynamic quench techniques. The time course of the forward reaction (22 degrees C) at pH 8.0 is characterized by a "burst" of AMP formation upon quenching with acid that is equivalent to 0.59 mol of enzyme. The pre-steady-state rate is followed by a slower steady-state rate of 0.60 s-1. The rate constant for the transient phase is 9.7 s-1. The time course for the formation of argininosuccinate is linear and shows neither a "lag" nor a burst phase. These results have been interpreted to mean that the mechanism for the formation of argininosuccinate consists of at least two distinct chemical steps with the formation of citrulline adenylate as a reactive intermediate. In the presence of aspartate the rate constant for the formation of citrulline adenylate (6.2 s-1) from ATP and citrulline is 7 times faster than the rate of formation of argininosuccinate from aspartate and citrulline adenylate (0.9 s-1). This suggests that the second step is predominantly rate limiting. The rate constant for the formation of citrulline adenylate in the absence of enzyme-bound aspartate (0.01 s-1) is 600 times slower than when aspartate is present. This indicates that the binding of aspartate to the enzyme regulates the formation of the intermediate. These results are in complete accord with our previously published steady-state kinetic scheme showing sequential addition of substrates.     

Hydrolytic action of aminoacyl-tRNA synthetases from baker's yeast. "Chemical proofreading" of Thr-tRNA Val by valyl-tRNA synthetase studied with modified tRNA Val and amino acid analogues.

The properties of native and of two modified tRNA Val species in the correction of misactivated threonine by valyl-tRNA synthetase have been studied. Whereas Thr-tRNA Val-C-C-A could not be isolated in the valyl-tRNA synthetase catalyzed reaction, Thr-tRNA Val-C-C-3'dA is isolable in up to 50% yield in this system and tRNA Val-C-C-3'NH2A is fully aminoacylated with threonine by the same enzyme. The hydrolysis of preformed Thr-tRNA Val-C-C-A by free valyl-tRNA synthetase is 30 times faster than the corresponding breakdown of Val-tRNA Val-C-C-A. This hydrolytic activity is also observed with Thr-tRNA Val-C-C-3'dA although the rate is reduce to that of the reaction of Val-tRNA Val-C-C-A. Modification of the threonine to O-methylthreonine, which is also a substrate for valyl-tRNA synthetase, leads to stabilization of the O-methylthreonyl-tRNA esters. The AMP/PP independent hydrolysis under aminoacylating conditions, which is a measure of the correction process, indicates that O-MeThr-tRNA Val-C-C-A is only very slowly corrected while the tRNA Val-C-C-3'dA and tRNA Val-C-C-3'NH2A esters are completely stable. Removal of the methoxy group of O-methylthreonine as in alpha-amino-butyric acid increases the rate of the hydrolytic reaction and once again alpha-Abu-tRNA Val-C-C-A and alpha-Abu-tRNA Val-C-C-3'dA are unstable under aminoacylating conditions and not isolable.     

Diffusion-dependent rates for the hydrolysis reaction catalyzed by glyoxalase II from rat erythrocytes

Glyoxalase II from rat erythrocytes is a near optimal catalyst for the hydrolysis of S-D-lactoylglutathione in the sense that the magnitude of kcat/Km is limited, in large part, by the rate constant for diffusion-controlled encounter between substrate and active site. The experimental basis for this conclusion is derived from the dependencies of the kinetic properties of the enzyme on solution viscosity (pH 7, Ic = 0.1 M, 25 degrees C). When sucrose is used as a viscogenic agent, kcat/Km for S-D-lactoylglutathione (8.8 x 10(5) M-1 s-1) decreases markedly with increasing solution viscosity. This effect appears not to be due to a sucrose-induced change in the intrinsic kinetic properties of the enzyme, since kcat/Km for the slow substrate S-acetylglutathione (3.7 x 10(4) M-1 s-1) is nearly independent of solution viscosity. Quantitative treatment of the data using Stoke's law indicates that the rate of hydrolysis of S-D-lactoylglutathione will be approximately 50% diffusion limited when [substrate] much less than Km; the encounter complex between enzyme and substrate partitions nearly equally between product formation and dissociation to form free enzyme and substrate. The same conclusion is reached when glycerol is used as a viscogenic agent, once the apparent activation effect of glycerol on the intrinsic activity of the enzyme is taken into account. Finally, the rate of formation of the encounter complex between substrate and active site may be governed to a significant extent by charge-charge interactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Achieving error-free translation; the mechanism of proofreading of threonyl-tRNA synthetase at atomic resolution

The fidelity of aminoacylation of tRNA(Thr) by the threonyl-tRNA synthetase (ThrRS) requires the discrimination of the cognate substrate threonine from the noncognate serine. Misacylation by serine is corrected in a proofreading or editing step. An editing site has been located 39 A away from the aminoacylation site. We report the crystal structures of this editing domain in its apo form and in complex with the serine product, and with two nonhydrolyzable analogs of potential substrates: the terminal tRNA adenosine charged with serine, and seryl adenylate. The structures show how serine is recognized, and threonine rejected, and provide the structural basis for the editing mechanism, a water-mediated hydrolysis of the mischarged tRNA. When the adenylate analog binds in the editing site, a phosphate oxygen takes the place of one of the catalytic water molecules, thereby blocking the reaction. This rules out a correction mechanism that would occur before the binding of the amino acid on the tRNA.     

Yellow lupin (Lupinus luteus) aminoacyl-tRNA synthetases. Isolation and some properties of enzyme-bound valyl adenylate and seryl adenylate.

As a continuation of our studies on plant (yellow lupin, Lupinus luteus) aminoacyl-tRNA synthetases we describe here formation and some properties of valyl-tRNA synthetase-bound valyl adenylate (EVal(Val-AMP)) and seryl-tRNA synthetase-bound seryl adenylate (ESer(Ser-AMP)). Valyl-tRNA synthetase-bound valyl adenylate was detected and isolated by several approaches in the pH range 6--10. In that range inorganic pyrophosphatase increases the amount of valyl adenylate by factor 1.8 regardless of pH. 50% of valine from the EVal(Val-AMP) complex isolated by Sephadex G-100 gel filtration was transferred to tRNA with a rate constant greater than 4 min-1 (pH 6.2, 10 degrees C). The ratio of valine to AMP in the enzyme-bound valyl adenylate is 1 : 1 and it is not changed by the presence of periodate-oxidized tRNA. In contrast to enzyme-bound valyl adenylate, formation of ESer(Ser-AMP) is very sensitive to pH. Inorganic pyrophosphatase increases the amount of seryl adenylate by a factor 6 at pH 8.0 and 30 at pH 6.9 60% of serine from the ESer(Ser-AMP) complex was transferred to tRNA with a rate constant greater than 4 min-1 (pH 8.0, 0 degrees C). The ratio of serine to AMP in the enzyme-bound seryl adenylate is 1 : 1. The rate of synthesis of the enzyme-bound aminoacyl adenylates was measured by ATP-PPi exchange. Michaelis constants for the substrates of valyl-tRNA and seryl-tRNA synthetases in ATP-PPi exchange were determined. Effects of pH, MgCl2 and KCl on the initial velocity of aminoacyl adenylate formation are described. For comparison, catalytic indices in the aminoacylation reactions catalyzed by both lupin enzymes are given and effects of pH, MgCl2 and KCl on tRNA aminoacylation are presented as well. Under some conditions, e.g. at low pH or high salt concentration, lupin valyl-tRNA and seryl-tRNA synthetase are active exclusively in ATP-PPi exchange reaction.     

Studies of the phosphoenzyme intermediate of the yeast plasma membrane proton-translocating ATPase

The yeast plasma membrane proton-pumping ATPase forms a phosphorylated intermediate during the hydrolysis of ATP. The fraction of enzyme phosphorylated during steady-state ATP hydrolysis was studied as a function of substrate concentration (MgATP), Mg2+ concentration, and pH. The dependence of the fraction of enzyme phosphorylated on the concentration of MgATP is sigmoidal, and the isotherms can be fit with parameters and mechanisms similar to those used to describe ATP hydrolysis. The isotherm is significantly more sigmoidal at pH 5.5 than at pH 6.0, with the limiting percentage (100.mol of phosphate/mol of enzyme) of enzyme phosphorylated being 70% and 6%, respectively, at the two pH values. The maxima in the steady-state rate of ATP hydrolysis occur at higher concentrations of Mg2+ and higher pH than the maxima in the fraction of enzyme phosphorylated. This suggests that the rate-determining step for ATP hydrolysis is different from that for enzyme phosphorylation and the hydrolysis of phosphoenzyme is enhanced by Mg2+ and high pH. The rate of phosphoenzyme formation was investigated with the quenched-flow method, but only a lower bound of 140 s-1 could be obtained for the rate constant at MgATP concentrations greater than 2.5 mM. Since the turnover number for ATP hydrolysis under similar conditions is 14 s-1, the rate-determining step in ATP hydrolysis occurs after enzyme phosphorylation.     

Sarcoplasmic reticulum adenosinetriphosphatase phosphorylation from inorganic phosphate. Theoretical and experimental reinvestigation

Pi phosphorylation of sarcoplasmic reticulum (SR) vesicles in the absence of Ca was reinvestigated. Theoretical analysis shows that, for various substrate concentrations, the time dependence of phosphoenzyme formation does not allow determination of an unambiguous reaction scheme or estimation of the stoichiometry of the reaction. To overcome this difficulty, we measured medium Pi oxygen exchange, [32P]-phosphoenzyme formation, and intrinsic fluorescence. We found that contrarily to the usual assumption the substrate binding step in the phosphorylation direction at pH 6.0, KCl = 0, and 23 degrees C is a slow process whose bimolecular rate constant is around 5 X 10(3) M-1 s-1 for both Mg and Pi binding. We confirm [Lacapère, J. J., Gingold, M. P., Champeil, P., & Guillain, F. (1981) J. Biol. Chem. 256, 2302-2306] that, in a second step, the establishment of a covalent bond between the bound Pi and the enzyme is formed with a rate constant greater than or equal to 20 s-1 whereas the dephosphorylation rate constant is 2-3 s-1. These results imply that under optimal conditions for phosphorylation, the enzyme is almost entirely phosphorylated at concentrations of 20 mM MgCl2 and 20 mM Pi. Study of the phosphorylation reaction under various experimental conditions shows that reduction of the phosphoenzyme level upon KCl addition is mainly due to the augmentation of the hydrolysis rate constant. In addition we propose that the strong inhibition by large amounts of MgCl2 is due to the formation of an E? . Mg complex unfit for phosphorylation by Pi.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pre-steady-state and steady-state kinetic analysis of the low molecular weight phosphotyrosyl protein phosphatase from bovine heart.

The complete time course of the hydrolysis of p-nitrophenyl phosphate catalyzed by the low molecular weight (acid) phosphotyrosyl protein phosphatase from bovine heart was elucidated and analyzed in detail. Burst titration kinetics were demonstrated for the first time with this class of enzyme. At pH 7.0, 4.5 degrees C, a transient pre-steady-state "burst" of p-nitrophenol was formed with a rate constant of 48 s-1. The burst was effectively stoichiometric and corresponded to a single enzyme active site/molecule. The burst was followed by a slow steady-state turnover of the phosphoenzyme intermediate with a rate constant of 1.2 s-1. Product inhibition studies indicated an ordered uni-bi kinetic scheme for the hydrolysis. Partition experiments conducted for several substrates revealed a constant product ratio. Vmax was constant for these substrates, and the overall rate of hydrolysis was increased greatly in the presence of alcohol acceptors. An enzyme-catalyzed 18O exchange between inorganic phosphate and water was detected and occurred with kcat = 4.47 x 10(-3) s-1 at pH 5.0, 37 degrees C. These results were all consistent with the existence of a phosphoenzyme intermediate in the catalytic pathway and with the breakdown of the intermediate being the rate-limiting step. The true Michaelis binding constant Ks = 6.0 mM, the apparent Km = 0.38 mM, and the rate constants for phosphorylation (k2 = 540 s-1) and dephosphorylation (k3 = 36.5 s-1) were determined under steady-state conditions with p-nitrophenyl phosphate at pH 5.0 and 37 degrees C in the presence of phosphate acceptors. The energies of activation for the enzyme-catalyzed hydrolysis at pH 5.0 and 7.0 were 13.6 and 14.1 kcal/mol, respectively. The activation energy for the enzyme-catalyzed medium 18O exchange between phosphate and water was 20.2 kcal/mol. Using the available equilibrium and rate constants, an energetic diagram was constructed for the enzyme-catalyzed reaction.     

Comparison of the processes involved in reduction by the substrate for two homologous flavocytochromes b2 from different species of yeast.

A detailed study of the electron exchanges involved between FMN and haem b2 groups within flavocytochrome b2 of yeast Hansenula anomala (H-enzyme) was performed. The results were compared with those for the homologous enzyme of yeast Saccharomyces cerevisiae (Sx-enzyme) re-investigated at 5 degrees C. The mid-point reduction potentials of FMN and haem were determined by two complementary methods: potentiometric titration with substrate, L-lactate, in the presence of dye mediators with quantification of the reduced species performed by spectrophotometry at suitable wavelengths; anaerobic titration of the enzyme by its substrate by monitoring the e.p.r. signals of the semiquinone and Fe3+ species. Values of Em,7 = -19, -23 and -45 V were determined respectively from the data for the three redox systems Ho/Hr, Fo/Fsq and Fsq/Fr in the H-enzyme instead of +6, -44 and -57 mV respectively in the Sx-enzyme [Capeillère-Blandin, Bray, Iwatsubo & Labeyrie (1975) Eur. J. Biochem. 54, 549-566]. Parallel e.p.r rapid-freezing and absorbance stopped-flow studies allowed determination of the time courses of the various redox species during their reduction by L-lactate. The flavin and the haem reduction time courses were biphasic. In the initial fast phase the reduction of flavin monitored by absorbance measurements is accomplished with a rate constant kF = 360 s-1. The reduction of the haem lags the reduction of flavin with a rate constant kH = 170 s-1. The appearance of flavin free radical is slower than the reduction in flavin absorbance and occurs with a rate constant close to that of the reduction of the haem. At saturating L-lactate concentration the initial rapid phase (up to 15 ms) involved in the overall turnover can be adequately simulated with a two-step reaction scheme. The main difference between the enzymes lies especially at the level of the first step of electron exchange between bound lactate and flavin, which for the H-enzyme is no longer the rate-limiting step in the haem reduction and becomes 8-fold faster than in the Sx-enzyme. Consequently in the H-enzyme for the following step, the intramolecular transfer from flavin hydroquinone to oxidized haem, a reliable evaluation of the rate constants becomes possible. Preliminary values are k+2 = 380 s-1 and k-2 = 120 s-1 at 5 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Kinetics of creatine phosphokinase and adenylate kinase. A two-dimensional NMR analysis

The kinetics of creatine phosphokinase and adenylate kinase catalyzed reactions were studied at equilibrium by two-dimensional Fourier transform phosphorus-31 nuclear magnetic resonance. For the creatine phosphokinase reaction, a pseudo-first-order rate constant of 0.29 s-1 was determined for the transfer of a phosphate group from adenosine triphosphate to creatine phosphate. For the adenylate kinase reaction two slow rate processes were required to describe the experimental results. The conversion of adenosine diphosphate to adenosine monophosphate was found to have a pseudo-first-order rate constant of 1.2 s-1, whereas that for the release of adenosine triphosphate from its enzyme complex occurred at a rate of 14 s-1.     

Kinetic and spectral properties of rabbit brain 4-aminobutyrate aminotransferase.

Purification and 4-aminobutyrate-2-oxoglutarate aminotransferase (EC 2.6.1.19) from rabbit brain is described. The method was used as a routine to give between 5 and 10mg of pure enzyme from 750 g of rabbit brain. The enzyme is a dimer made up of subunits each with a mol. wt. of 58000. An absorption spectrum of the freshly prepared enzyme shows peaks at 415 and 330 nm. Treatment of the enzyme with the substrate 4-amino-butyrate or glutamate produces a decrease in the 415 nm and an increase in the 330 nm peak. This conversion, which is attributed to an aldimine into ketimine step in the reaction, is sufficiently slow when 4-aminobutyrate is the substrate to allow it to be followed by stopped-flow spectrophotometry. A first-order rate constant was determined for this step (12s-1) and compared with the turnover number for the enzyme derived by steady-state methods (9.5S-1). The first-order rate constant when glutamate was used as substrate was estimated to be approx. 30s-1.     

Activation of Escherichia coli pyruvate oxidase enhances the oxidation of hydroxyethylthiamin pyrophosphate

The catalytic efficiency (kcat/Km) of Escherichia coli flavin pyruvate oxidase can be stimulated 450-fold either by the addition of lipid activators or by limited proteolytic hydrolysis. Previous studies have shown that a functional lipid binding site is a mandatory prerequisite for the in vivo functioning of this enzyme (Grabau, C., and Cronan, J. E., Jr. (1986) Biochemistry 25, 3748-3751). The effect of activation on the transient state kinetics of partial reactions in the overall oxidative conversion of pyruvate to acetate and CO2 has now been examined. The rate of decarboxylation of pyruvate to form CO2 and hydroxyethylthiamin pyrophosphate for both activated and unactivated forms of the enzyme is identical within experimental error. The decarboxylation step was measured using substrate concentrations of the enzyme in the absence of an electron acceptor. The pseudo-first order rate constant for the decarboxylation step is 60-80 s-1. The rate of oxidation of hydroxyethylthiamin pyrophosphate and concomitant enzyme-bound flavin reduction was analyzed by stopped-flow methods utilizing synthetic hydroxyethylthiamin pyrophosphate. The pseudo-first order rate for this step with unactivated enzyme was 2.85 s-1 and increased 145-fold for lipid-activated enzyme to 413 s-1 and 61-fold for the proteolytically activated enzyme to 173 s-1. The analysis of a third reaction step, the reoxidation of enzyme-bound FADH, was also investigated by stopped-flow techniques utilizing ferricyanide as the electron acceptor. The rate of oxidation of enzyme.FADH is very fast for both unactivated (1041 s-1) and activated enzyme (645 s-1). The data indicate that the FAD reduction step is the rate-limiting step in the overall reaction for unactivated enzyme. Alternatively, the rate-limiting step in the overall reaction with the activated enzyme shifts to one of the partial steps in the decarboxylation reaction.     

Conformational movements and cooperativity upon amino acid,ATP and tRNA binding in threonyl-tRNA synthetase

The crystal structures of threonyl-tRNA synthetase (ThrRS) from Staphylococcus aureus, with ATP and an analogue of threonyl adenylate, are described. Together with the previously determined structures of Escherichia coli ThrRS with different substrates, they allow a comprehensive analysis of the effect of binding of all the substrates: threonine, ATP and tRNA. The tRNA, by inserting its acceptor arm between the N-terminal domain and the catalytic domain, causes a large rotation of the former. Within the catalytic domain, four regions surrounding the active site display significant conformational changes upon binding of the different substrates. The binding of threonine induces the movement of as much as 50 consecutive amino acid residues. The binding of ATP triggers a displacement, as large as 8A at some C(alpha) positions, of a strand-loop-strand region of the core beta-sheet. Two other regions move in a cooperative way upon binding of threonine or ATP: the motif 2 loop, which plays an essential role in the first step of the aminoacylation reaction, and the ordering loop, which closes on the active site cavity when the substrates are in place. The tRNA interacts with all four mobile regions, several residues initially bound to threonine or ATP switching to a position in which they can contact the tRNA. Three such conformational switches could be identified, each of them in a different mobile region. The structural analysis suggests that, while the small substrates can bind in any order, they must be in place before productive tRNA binding can occur.     

Kinetics of pump currents generated by the Na+,K+-ATPase

Purified Na+,K+-ATPase from pig kidney was attached to black lipid membranes. Pump currents of the enzyme could be measured with a time resolution of approx. 1 ms by releasing ATP from caged ATP with a UV laser flash. Analysis of the transient currents shows that a slow non-electrogenic step is followed by an electrogenic transition with a rate constant of 100 s-1 (22 degrees C). The exponential components found in the transient currents are compared to transitions in the Albers-Post scheme.     

Mutational separation of two pathways for editing by a class I tRNA synthetase

Aminoacyl tRNA synthetases (aaRSs) catalyze the first step in protein biosynthesis, establishing a connection between codons and amino acids. To maintain accuracy, aaRSs have evolved a second active site that eliminates noncognate amino acids. Isoleucyl tRNA synthetase edits valine by two tRNA(Ile)-dependent pathways: hydrolysis of valyl adenylate (Val-AMP, pretransfer editing) and hydrolysis of mischarged Val-tRNA(Ile) (posttransfer editing). Not understood is how a single editing site processes two distinct substrates--an adenylate and an aminoacyl tRNA ester. We report here distinct mutations within the center for editing that alter adenylate but not aminoacyl ester hydrolysis, and vice versa. These results are consistent with a molecular model that shows that the single editing active site contains two valyl binding pockets, one specific for each substrate.     

The aminopeptidase from Aeromonas proteolytica can function as an esterase

The aminopeptidase from Aeromonas proteolytica (AAP) can catalyze the hydrolysis of L-leucine ethyl ester ( L-Leu-OEt) with a rate of 96 +/- 5 s-1 and a Km of 700 microM. The observed turnover number for L-Leu-OEt hydrolysis by AAP is similar to that observed for peptide hydrolysis, which is 67 +/- 5 s-1. The k(cat) values for the hydrolysis of L-Leu-OEt and L-leucine- p-nitroanilide ( L- pNA) catalyzed by AAP were determined at different pH values under saturating substrate concentrations. Construction of an Arrhenius plot from the temperature dependence of AAP-catalyzed ester hydrolysis indicates that the rate-limiting step does not change as a function of temperature and is product formation. The activation energy ( Ea) for the activated ES ester complex is 13.7 kJ mol-1, while the enthalpy and entropy of activation at 25 degrees C calculated over the temperature range 298-338 K are 11.2 kJ mol-1 and -175 J K-1 mol-1, respectively. The free energy of activation at 25 degrees C was found to be 63.4 kJ mol-1. The enthalpy of ionization was also measured and was found to be very similar for both peptide and ester substrates, yielding values of 20 kJ mol-1 for L-Leu-OEt and 25 kJ mol-1 for L- pNA. For peptide and L-amino acid ester cleavage reactions catalyzed by AAP, and 6.07, respectively. Proton inventory data suggest that two protons are transferred in the rate-limiting step of ester hydrolysis while only one is transferred in peptide hydrolysis. The combination of these data with the available X-ray crystallographic, kinetic, spectroscopic, and thermodynamic data for AAP provides new insight into the catalytic mechanism of AAP.     

Kinetic analysis of the hydrolysis of GTP by p21N-ras. The basal GTPase mechanism

The rate constants have been determined for elementary steps in the basal GTPase mechanism of normal p21N-ras (Gly-12) and an oncogenic mutant (Asp-12): namely GTP binding, hydrolysis, phosphate release, and GDP release. By extrapolation from data at lower temperatures, the GTP association rate constant at 37 degrees C is 1.4 x 10(8) M-1 s-1 for the normal protein and 4.8 x 10(8) M-1 s-1 for the mutant. Other rate constants were measured directly at 37 degrees C, and three processes have similar slow values. GTP dissociation is at 1.0 x 10(-4) s-1 (normal) and 5.0 x 10(-4) s-1 (mutant). The hydrolysis step is at 3.4 x 10(-4) s-1 (normal) and 1.5 x 10(-4) s-1 (mutant). GDP dissociates at 4.2 x 10(-4) s-1 (normal) and 2.0 x 10(-4) s-1 (mutant). GDP association rate constants are similar to those for GTP, 0.5 x 10(8) M-1 s-1 for normal and 0.7 x 10(8) M-1 s-1 for mutant. Both hydrolysis and GDP release therefore contribute to rate limitation of the basal GTPase activity. There are distinct differences (up to 5-fold) between rate constants for the normal and mutant proteins at a number of steps. The values are consistent with the reduced GTPase activity for this mutant and suggest little difference between normal and mutant proteins in the relative steady-state concentrations of GTP and GDP complexes that may represent active and inactive states. The results are discussed in terms of the likely role of p21ras in transmembrane signalling.     

Cytochrome oxidase from Pseudomonas aeruginosa. IV. Reaction with oxygen and carbon monoxide.

The reaction between a cytochrome oxidase from Pseudomonas aeruginosa and oxygen has been studied by a rapid mixing technique. The data indicate that the heme d1 moiety of the ascorbate-reduced enzyme is oxidized faster than the heme c component. The oxidation of heme d1 is accurately second order with respect to oxygen and has a rate constant of 5.7 - 10(4) M-1 - s-1 at 20 degrees C. The oxidation of the heme c has a first order rate constant of about 8 s-1 at infinite concentration of O2. The results indicate that the rate-limiting step is the internal transfer of electrons from heme c to heme d1. These more rapid reactions are followed by more complicated but smaller abcorbance changes whose origin is still not clear. The reaction of ascorbate-reduced oxidase with CO has also been studied and is second order with a rate constant of 1.8 - 10(4) M-1 - s-1. The initial reaction with CO is followed by a slower reaction of significantly less magnitude. The equilibrium constant for the reaction with CO, calculated as a dissociation constant from titrimetric experiments with dithionite-reduced oxidase, is about 2.3 - 10(-6) M. From these data a rate constant of 0.041 s-1 can be calculated for the dissociation of CO from the enzyme.