Yeast seryl tRNA synthetase: two sets of substrate sites involved in aminoacylation.

Seryl tRNA synthetase from Saccharomyces Carlsbergensis C836 contains two sets of sites for tRNASer, L-serine, and Mg2+-ATP, both of which are involved in aminoacylation. This is based on the following experimental results: (a) at low serine concentrations, second order kinetics in tRNASer are observed; (b) biphasic kinetics result when the amino acid is the varied substrate indicating anticooperative binding of two serine molecules to the synthetase; (c) when two molecules of serine are bound the rate of aminoacylation increases strongly and becomes first order in tRNASer; (d) the involvement of more than one site for Mg2+ and ATP is deduced from systematic variations of the concentrations of Mg2+ and ATP. Implications of the anticooperative binding of the substrates for possible reaction mechanisms are discussed. The results indicate that under normal conditions, the activity of seryl tRNA synthetase is regulated mainly by tRNASer while at high serine concentrations regulation by the amino acid itself prevails.     

Yeast seryl tRNA synthetase: interactions between the ATP binding site and the sites for tRNASer and L-serine.

T1 ribonuclease digestion of yeast tRNASer in the presence of seryl tRNA synthetase was used for monitoring the relationship between the substrate binding sites on the synthetase. It was found that (a) ATP displaces the tRNA from the synthetase with an effector affinity constant corresponding to the Km for ATP of 10 micron; (b) AMP and a number of nucleoside triphosphates, while influencing the rate of aminoacylation, do not displace the tRNA from the enzyme; (c) ADP and PPi inhibit the aminoacylation and the binding of tRNASer; (d) adenylyl diphosphonate is bound to the synthetase and lowers the protection of the tRNA against the nuclease attack in a similar way as does ATP; (e) interactions between the sites of L-serine and tRNASer could only be shown when both sites for serine were saturated and, in addition, the ATP analog or ADP was present. It is concluded that in seryl tRNA synthetase binding sites for ATP interact with the ones for tRNA as well as with the ones for serine. These findings contribute to the understanding of the mechanism of aminoacylation.     

Unusual transient- and steady-state kinetic behavior is predicted by the kinetic scheme operational for recombinant human dihydrofolate reductase

Association and dissociation rate constants obtained by stopped-flow spectroscopy have permitted definition of a kinetic scheme for recombinant human dihydrofolate reductase that correctly predicts full time course kinetics of the enzymatic reaction over a wide range of substrate and product concentrations. The scheme is complex compared with that for the bacterial enzyme and involves branched pathways. It successfully accounts for observed rapid hysteresis preceding steady state and for the nonhyperbolic dependence of steady-state rate on substrate and product concentrations. The major branch point in the catalytic cycle occurs at E.NADP.H4folate because either NADP or H4folate can dissociate from the ternary product complex (koff = 84 s-1 and 46 s-1, respectively). The rate of conversion of enzyme-bound substrates to products is very fast (k = 1360 s-1) and nearly unidirectional (Kequ = 37) so that other steps limit the catalytic rate. At saturating substrate concentrations these steps include release of NADP and H4folate from E.NADP.H4folate and release of products from the two abortive complexes E.NADPH.H4folate (koff = 225 s-1) and E.NADP.H4folate (koff = 4.6 s-1). Since NADP dissociates slowly from E.NADP.H2folate nearly 90% of the enzyme accumulates as this complex at steady state. Nonetheless, the catalytic rate is maintained at 12 s-1 by rapid flux of a small portion of the enzyme through an alternate branch. At physiological concentrations of substrates and products the steady-state rate is limited primarily by the rate of H2folate binding to E.NADPH so that the enzyme is extremely efficient.     

Transient kinetic studies on the allosteric transition of phosphoglycerate dehydrogenase.

Stopped flow spectrophotometry was used to investigate the kinetics of the transition of the phosphoglycerate dehydrogenase (3-phosphoglycerate: NAD oxidoreductase, EC 1.1.1.95) reaction from the active to the inhibited rate upon the addition of the physiological inhibitor serine. The transition was characterized by a single first order rate constant (kobs,i) which was independent of enzyme concentration. At pH 8.5, kobs,i increased in a hyperbolic manner with serine concentration from 2 to 8 s-1. The increase in kobs,i occurred at serine concentrations where the steady state inhibition was virtually complete. These results indicate that serine inhibition is an allosteric process involving a conformational change in the enzyme. A model is presented in which serine at low concentrations binds exclusively to the inhibited state of the enzyme and shifts the equilibrium toward that state; at high serine concentrations, serine binds to the active state, facilitating its conversion to the inhibited state. An alternative model, which we favor, proposes two classes of inhibitor binding sites. The kinetics of the fluorescence quenching of enzyme-bound NADH by serine (Sugimoto, E., and Pizer, L.I. (1968) J. Biol. Chem. 243, 2090-2098), measured by stopped flow fluorimetry, was also characterized by a single first order rate constant (kobs,f.q.) which was independent of enzyme concentration. At pH 8.5, kobs,f.q. ranged from 0.4 s-1 at low serine concentrations to 1.1 s-1 at high serine concentrations. These results indicate that the fluorescence quenching induced by serine is a manifestation of a structural change in the enzyme. Enzyme and excess NADH were mixed with substrate and serine in the stopped flow instrument, and enzyme-bound NADH fluorescence was monitored by exciting through the protein at 285 nm. A rapid fluorescence quenching process, which occurred within the mixing time, was followed by a slower fluorescence enhancement process which terminated in a steady state level corresponding to the quenched fluorescence of the enzyme NADH serine complex. The rapid quenching was the result of substrate binding (Dubrow, R., and Pizer, L.I. (1977) J. Biol. Chem. 252, 1539-1551). The fluorescence enhancement was characterized by a single first order rate constant whose value for a given serine concentration corresponded with Kobs,j. This data shows that the quenched state of the enzyme-NADH-complex is the state which is directly responsible for the inhibition of enzyme activity. During catalysis the quenched state is achieved from a different initial conformation, and consequently at a different rate, than in the absence of substrate. kobs,j and kobs,f.q. were also measured using glycine, another inhibitor. The ultraviolet difference spectrum between enzyme and enzyme plus serine was determined and proposed to be the result of the same structural change which is responsible for the fluorescence quenching by serine.     

Direct measurement of metal ion chelation in the active site of human ferrochelatase

The final step in heme biosynthesis, insertion of ferrous iron into protoporphyrin IX, is catalyzed by protoporphyrin IX ferrochelatase (EC 4.99.1.1). We demonstrate that pre-steady state human ferrochelatase (R115L) exhibits a stoichiometric burst of product formation and substrate consumption, consistent with a rate-determining step following metal ion chelation. Detailed analysis shows that chelation requires at least two steps, rapid binding followed by a slower (k approximately 1 s-1) irreversible step, provisionally assigned to metal ion chelation. Comparison with steady state data reveals that the rate-determining step in the overall reaction, conversion of free porphyrin to free metalloporphyrin, occurs after chelation and is most probably product release. We have measured rate constants for significant steps on the enzyme and demonstrate that metal ion chelation, with a rate constant of 0.96 s-1, is approximately 10 times faster than the rate-determining step in the steady state (kcat = 0.1 s-1). The effect of an additional E343D mutation is apparent at multiple stages in the reaction cycle with a 7-fold decrease in kcat and a 3-fold decrease in kchel. This conservative mutation primarily affects events occurring after metal ion chelation. Further evaluation of structure-function data on site-directed mutants will therefore require both steady state and pre-steady state approaches.     

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.     

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.     

Relationship of stopped flow to steady state parameters in the dimeric copper amine oxidase from Hansenula polymorpha and the role of zinc in inhibiting activity at alternate copper-containing subunits

The expression of a copper amine oxidase (CAO) from Hansenula polymorpha in Saccharomyces cerevisiae under differing culture conditions leads to the incorporation of varied levels of CAO-bound zinc. The presence of substantial amount of zinc results in two distinctive enzyme species, designated as the fast and slow enzymes. Both forms are rapidly reduced by substrate methylamine with a rate constant of 199 s(-1) but behave remarkably differently in their oxidation rates; the fast enzyme is oxidized by dioxygen at a rate of 22.1 s(-1), whereas the slow enzyme reacts at a rate of 1.8 x 10(-4) s(-1). The apparent kcat of the enzyme preparation is linearly proportional to the fraction of the fast enzyme, with an extrapolated value of 6.17 s(-1) when the enzyme is 100% in its "fast" form. A comparison of rate constants for cofactor reduction and reoxidation steps, measured in stopped flow experiments, to the extrapolated kcat implicates additional steps in the steady state reaction. Measurement of the proportion of oxidized (ETPQ(ox)) and reduced cofactor (ETPQ(red)) under steady state conditions indicates approximately 50% of each cofactor form at 0.8 or 2 mM methylamine. Kinetic isotope effect measurements using deuterated amine substrate lead to the following steady state values: (D)(k(red)) = 8.5 (0.5), (D)(kcat) = 1.7 (0.1), and (D)(kcat/K(m)) = 4.3 (0.2). The collective data allow the calculation of partially rate-determining constants during the reductive half-reaction (ca. 200 s(-1) for binding of substrate to ETPQ(ox) and 27.9 s(-1) for release of aldehyde product or a protein isomerization from ETPQ(red)); an additional step with a rate constant of 13.2 s(-1) is assigned to the oxidative half-reaction, most likely for the release of product hydrogen peroxide. These results, together with the sole detection of oxidized and reduced cofactor during rapid scanning stopped flow experiments, indicate that four steps contribute to kcat, with the first electron transfer from cofactor to O2 contributing ca. 29%. An investigation of the relationship between the copper content and the extent of the fast enzyme shows that only the copper-containing homodimer is capable of rapid reoxidation and that zinc-copper heterodimers are incapable of rapid turnover at either subunit. This implies communication between the metal sites of the two subunits per dimer that impacts O2 binding and/or electron transfer from reduced cofactor to bound O2.     

Tryptophanyl adenylate formation by tryptophanyl-tRNA synthetase from Escherichia coli

By gel filtration and titration on DEAE-cellulose filters we show that Escherichia coli tryptophanyl-tRNA synthetase forms tryptophanyl adenylate as an initial reaction product when the enzyme is mixed with ATP-Mg and tryptophan. This reaction precedes the synthesis of the tryptophanyl-ATP ester known to be formed by this enzyme. The stoichiometry of tryptophanyl adenylate synthesis is 2 mol per mole of dimeric enzyme. When this reaction is studied either by the stopped-flow method, by the fluorescence changes of the enzyme, or by radioactive ATP depletion, three successive chemical processes are identified. The first two processes correspond to the synthesis of the two adenylates, at very different rates. The rate constants of tryptophanyl adenylate synthesis are respectively 146 +/- 17 s-1 and 3.3 +/- 0.9 s-1. The third process is the synthesis of tryptophanyl-ATP, the rate constant of which is 0.025 s-1. The Michaelis constants for ATP and for tryptophan in the activation reaction are respectively 179 +/- 35 microM and 23.9 +/- 7.9 microM, for the fast site, and 116 +/- 45 microM and 3.7 +/- 2.2 microM, for the slow site. No synergy between ATP and tryptophan can be evidenced. The data are interpreted as showing positive cooperativity between the subunits associated with conformational changes evidenced by fluorometric methods. The pyrophosphorolysis of tryptophanyl adenylate presents a Michaelian behavior for both sites, and the rate constant of the reverse reaction is 360 +/- 10 s-1 with a binding constant of 196 +/- 12 microM for inorganic pyrophosphate (PPi).(ABSTRACT TRUNCATED AT 250 WORDS)     

Transient kinetic and deuterium isotope effect studies on the catalytic mechanism of phosphoglycerate dehydrogenase.

The catalytic mechanism of the phosphoglycerate dehydrogenase reaction in both directions was investigated by studying: (a) pre-steady state transients in reduced coenzyme appearance or disappearance or disappearance and in protein fluorescence; (b) deuterium isotope effects on the transients and on the steady state reactions; and (c) the partial reaction between the enzyme-NADH complex and hydroxypyruvate-P. These studies led to the scheme below for the ternary complex interconversion. E1-NADH-hydroxypyruvate-P(1)equilibriumE2-NADH-hydroxypyruvate-P(2)equilibriumE3-NADH-hydroxypyruvate-P + H+(3)equilibriumE3-NAD+-3-phosphoglycerate(4)equilibriumE4-NAD+-3-phosphoglycerate Steps 1,2, and 4 are ternary complex isomerizations. Step 3 is the hydride transfer. Under steady state conditions isomerization 2 is the rate-determining step in the direction of hydroxypyruvate-P reduction at higher pH values. At lower pH values, the hydride transfer step is also partially rate-determining. The rate-determining step in the direction of 3-phosphoglycerate oxidation occurs subsequent to the hydride transfer step at higher pH values. At lower pH values the rate is determined by both isomerization 4 and the hydride transfer step. Isomerizations 1, 2, and 4 were inhibited by serine, an allosteric inhibitor, indicating that the inactive conformation of the enzyme is incapable of performing any of the steps of the ternary complex interconversion. Phosphoglycerate dehydrogenase corresponds to a V-type allosteric enzyme. When the enzyme-NADH complex was mixed with hydroxypyruvate-P at pH 8.5, a rapid quenching of enzymebound NADH fluorescence occurred. This process was studied under pseudo-first order conditions and shown to be the result of hydroxypyruvate-P binding.     

Origins of reversed directionality in the ncd molecular motor.

The head or motor domain of the ncd (non-claret disjunctional) molecular motor is 41% identical to that of kinesin, yet moves along microtubules in the opposite direction to kinesin. We show here that despite the reversed directionality of ncd, its kinetics in solution are homologous in key respects to those of kinesin. The rate limiting step, ADP release, occurs at 0.0033 s-1 at 100 mM NaCl and is accelerated approximately 1000-fold when the motor binds to microtubules. Other reaction steps are all very fast (> 0.1 s-1) compared with ADP release, and the motor is consequently paused in the ncd.ADP state until microtubule binding occurs (Kd = 2 microM), at which point ADP release is triggered and the motor locks onto the microtubule in a rigor-like state. These data identify close functional homology between the strong binding states of kinesin and ncd, and in view of this we discuss a possible mechanism for directional reversal, in which the strong binding states of ncd and kinesin are functionally identical, but the weak binding states are biased in opposite directions.     

Elementary steps of the actin-activated ATPase reaction of cardiac muscle myosin subfragment-1

The rates of the elementary steps of the actomyosin ATPase reaction were measured using the myosin subfragment-1 of porcine left ventricular muscle. The results could be explained only by the two-route mechanism for actomyosin ATPase (Inoue, Shigekawa, & Tonomura (1973) J. Biochem. 74, 923-934), in which ATP is hydrolyzed via routes with or without accompanying dissociation of actomyosin. The dependence on the F-actin concentration of the rate of the acto-S-1 ATPase reaction in the steady state was measured in 5 mM KCl at 20 degrees C. The maximal rate, Vmax, and the dissociation constant for F-actin of the ATPase, Kd, were 3.0 s-1 and 2.2 mg/ml, respectively. The Kd value was almost the same as that determined from the extent of binding of S-1 with F-actin during the ATPase reaction. The rate of recombination of the S-1-phosphate-ADP complex, S-1ADPP, with F-actin, vr, was lower than that of the ATPase reaction in the steady state. Thus, ATP is mainly hydrolyzed without accompanying dissociation of acto-S-1 into S-1ADPP and F-actin. In the cardiac acto-S-1 ATPase reaction, the rate of the ATPase reaction in the steady state and that of recombination of S-1ADPP with F-actin were about 1/5 those of the skeletal acto-S-1 ATPase reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of 31P NMR spectra of enzyme-bound reactants and products of adenylate kinase using density matrix theory of chemical exchange

31P NMR spectra of equilibrium mixtures of enzyme-bound reactants and products of the adenylate kinase reaction (formula; see text) were analyzed by using computer simulations based on density matrix theory of chemical exchange. Since adenylate kinase has the unique feature that the reactants in the reverse direction are both ADP molecules, which are indistinguishable off the enzyme, the density matrix equations are formulated for the ABC + D in equilibrium A'B' + A"B" exchange appropriate for the reaction, in which the interchange of A'B' and A"B" is explicitly introduced. It is shown that the consideration of this interchange is essential to explain the experimentally observed line shapes. By comparison of the computer-simulated spectra with various values for the rates of the exchange with the experimental spectra for porcine adenylate kinase at pH 7.0 and T = 4 degrees C, the following characteristic rates were determined: interconversion rates, 375 +/- 30 s-1 (ATP formation) and 600 +/- 50 s-1 (ADP formation); interchange rates of donor and acceptor ADP's, 100 +/- 30 s-1 (in the presence of optimal Mg2+ concentration), 1500 +/- 100 s-1 (in the absence of Mg2+). It is shown that under the conditions of the experiments the interchange rate is the lower limit of the dissociation rate of ADP (or MgADP from the acceptor site if Mg2+ was present) from the enzyme complexes. The significance of these interchange rates and their values relative to the interconversion rates is discussed with special reference to the role of the Mg2+ ion in the differentiation of the two nucleotide binding sites on adenylate kinase.     

Inhibition of phosphoglucomutase by vanadate

Phosphoglucomutase is inhibited by a complex formed from alpha-D-glucose 1-phosphate (Glc-1-P) and inorganic vanadate (Vi). Both the inhibition at steady state and the rate of approach to steady state are dependent on the concentrations of both Glc-1-P and Vi. Inhibition is competitive versus alpha-D-glucose 1,6-bisphosphate (Glc-P2) and is ascribed to binding of the 6-vanadate ester of Glc-1-P (V-6-Glc-1-P) to the dephospho form of phosphoglucomutase (E). The inhibition constant for V-6-Glc-1-P at pH 7.4 was determined from steady-state kinetic measurements to be 2 x 10(-12) M. The first-order rate constant for approach to steady state increases hyperbolically with inhibitor concentration. The results are consistent with rapid equilibrium binding of V-6-Glc-1-P to E, with dissociation constant 1 x 10(-9) M, followed by rate-limiting conversion of the E.V-6-Glc-1-P complex to another species, E*.V-6-Glc-1-P, with first-order rate constant 4 x 10(-2)s-1. The rate constant determined for the reverse reaction, conversion of E*.V-6-Glc-1-P to E.V-6-Glc-1-P, is 2.5 x 10(-4)s-1. Formation of E*.V-6-Glc-1-P can also occur via binding of glucose 6-vanadate to the phospho form of phosphoglucomutase (E-P) followed by phosphoryl transfer and rearrangement of the enzyme-product complex.     

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.     

Enzyme hyperspecificity. Rejection of threonine by the valyl-tRNA synthetase by misacylation and hydrolytic editing.

Valyl-tRNA synthetase from Bacillus stearothermophilus activates thereonine and forms a 1:1 complex with threonyl adenylate, but it does not catalyze the net formation of threonyl-tRNAVal at pH 7.78 and 25 degrees C in the quenched flow apparatus it decomposes at a rate constant of 36s-1. During this process there is a transient formation of Thr-tRNAVal reaching a maximum at 25 ms and rapidly falling to zero after 150 ms. At the peak, 22% of the (14C) threonine from the complex is present as (14C) Thr-tRNA. The reaction may be quenched with phenol and the partially mischarged tRNA isolated. The enzyme catalyzes its hydrolysis with a rate constant of 40s-1. The data fit a kinetic scheme in which 62% of the threonine from the threonyl adenylate is transferred to the tRNA. This may be compared with the rate constant of 12s-1 at which 84% of the valine is transferred to tRNAVal from the enzyme-bound valyl adenylate, and the rate constant of 0.015s-1 for the subsequent hydrolysis of Val-tRNAVal. Inhibition studies indicate a distinct second site for hydrolysis. The translocation of the aminoacyl moiety between the two sites could be mediated by a transfer between the 2'-and 3'-OH groups of the terminal adenosine fo the tRNA. The hyperspecificity of the enzyme is based on discriminating between the two competing substrates twice: once against the undesired substrate in the synthetic step, and once against the desired substrate in the destructive step.     

tRNA-dependent amino acid discrimination by yeast seryl-tRNA synthetase.

The ability of aminoacyl-tRNA synthetases to distinguish between similar amino acids is crucial for accurate translation of the genetic code. Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS) employs tRNA-dependent recognition of its cognate amino acid serine [Lenhard, B., Filipic, S., Landeka, I., Skrtic, I., S?ll, D. & Weygand-Durasevic, I. (1997) J. Biol. Chem.272, 1136-1141]. Here we show that dimeric SerRS enzyme complexed with one molecule of tRNASer is more specific and more efficient in catalyzing seryl-adenylate formation than the apoenzyme alone. Sequence-specific tRNA-protein interactions enhance discrimination of the amino acid substrate by yeast SerRS and diminish the misactivation of the structurally similar noncognate threonine. This may proceed via a tRNA-induced conformational change in the enzyme's active site. The 3'-terminal adenosine of tRNASer is not important in effecting the rearrangement of the serine binding site. Our results do not provide an indication for a readjustment of ATP binding in a tRNA-assisted manner. The stoichiometric analyses of the complexes between the enzyme and tRNASer revealed that two cognate tRNA molecules can be bound to dimeric SerRS, however, with very different affinities.     

Serine modulates substrate channeling in tryptophan synthase. A novel intersubunit triggering mechanism

Tryptophan synthase, an alpha 2 beta 2 complex, is a classic example of an enzyme that is thought to "channel" a metabolic intermediate (indole) from the active site of the alpha subunit to the active site of the beta subunit. We now examine the kinetics of substrate channeling by tryptophan synthase directly by chemical quench-flow and stopped-flow methods. The conversion of indole-3-glycerol phosphate (IGP) to tryptophan at the active site proceeds at a rate of 24 s-1, which is limited by the rate of cleavage of IGP to produce indole (alpha reaction). In a single turnover experiment monitoring the conversion of radiolabeled IGP to tryptophan, only a trace of indole is detectable (less than or equal to 1% of the IGP), implying that the reaction of indole to form tryptophan must be quite fast (greater than or equal to 1000 s-1). The rate of reaction of indole from solution is much too slow (40 s-1 under identical conditions) to account for the negligible accumulation of indole in a single turnover. Therefore, the indole produced at the alpha site must be rapidly channeled to the beta site, where it reacts with serine to form tryptophan: channeling and the reaction of indole to form tryptophan must each occur at rates greater than or equal to 1000 s-1. Steady-state turnover is limited by the slow rate of tryptophan release (8 s-1). In the absence of serine, the cleavage of IGP to indole is limited by a change in protein conformation at a rate of 0.16 s-1. When the alpha beta reaction is initiated by mixing enzyme with IGP and serine simultaneously, there is a lag in the cleavage IGP and formation of tryptophan. The kinetics of the lag correspond to the rate of formation of the aminoacrylate in the reaction of serine with pyridoxal phosphate at the beta site, measured by stopped-flow methods (45 s-1). There is also a change in protein fluorescence, suggestive of a change in protein conformation, occurring at the same rate. Substitution of cysteine for serine leads to a longer lag in the kinetics of IGP cleavage and a correspondingly slower rate of formation of the aminoacrylate (6 s-1). Thus, the reaction of serine at the beta site modulates the alpha reaction such that the formation of the aminoacrylate leads to a change in protein conformation that is transmitted to the alpha site to enhance the rate of IGP cleavage 150-fold.(ABSTRACT TRUNCATED AT 400 WORDS)     

Binding of ADP and orthophosphate during the ATPase reaction of nitrogenase

The pre-steady-state ATPase activity of nitrogenase from Azotobacter vinelandii was investigated. By using a rapid-quench technique, it has been demonstrated that with the oxidized nitrogenase complex the same burst reaction of MgATP hydrolysis occurs as observed with the reduced complex, namely 6-8 mol orthophosphate released/mol MoFe protein. It is concluded that the pre-steady-state ATPase activity is independent of electron transfer from Fe protein to MoFe protein. Results obtained from gel centrifugation experiments showed that during the steady state of reductant-independent ATP hydrolysis there is a slow dissociation of one molecule of MgADP from the nitrogenase proteins (koff less than or equal to 0.2 s-1); the second MgADP molecule dissociates much faster (koff greater than or equal to 0.6 s-1). Under the same conditions orthophosphate was found to be associated with the nitrogenase proteins. The rate of dissociation of orthophosphate from the nitrogenase complex, as estimated from the gel centrifugation experiments, is in the same order of magnitude as the steady-state turnover rate of the reductant-independent ATPase activity (0.6 mol Pi formed X s-1 X mol Av2(-1) at 22 degrees C). These data are consistent with dissociation of orthophosphate or MgADP being rate-limiting during nitrogenase-catalyzed reductant-independent ATP hydrolysis.