Isoleucyl-tRNA synthetase from Baker's yeast. Catalytic mechanism, 2',3'-specificity and fidelity in aminoacylation of tRNAIle with isoleucine and valine investigated with initial-rate kinetics using analogs of tRNA, ATP and amino acids

The aminoacylation of three modified tRNAIle species with isoleucine and with valine by isoleucyl-tRNA synthetase has been investigated by initial rate kinetics. For aminoacylation of tRNAIle-C-C-3'dA with isoleucine, a bi-bi uni-uni ping-pong mechanism has been found by bisubstrate kinetics and inhibition by products and by 3'dATP; for aminoacylation with valine a bi-uni uni-bi ping-pong mechanism. For isoleucylation of tRNAIle-C-C-A(3'NH2) bisubstrate kinetics, inhibition by products and by isoleucinol show a random uni-bi uni-uni-uni ping-pong mechanism; for valylation of this tRNA a bi-bi uni-uni ping-pong mechanism is observed by bisubstrate kinetics and product inhibition. tRNAIle-C-C-2'dA was aminoacylated under modified conditions with isoleucine in a bi-bi uni-uni ping-pong mechanism with a rapid equilibrium segment as observed by bisubstrate kinetics, inhibition by AMP, by P[NH]P as product analog and by isoleucinol. Aminoacylation with valine is achieved in a rapid-equilibrium sequential random AB, ordered C mechanism indicated by bisubstrate kinetics and inhibition by 3'dATP and valinol. All six reactions exhibit orders of substrate addition and product release which are different from those observed in aminoacylation of the natural tRNAIle-C-C-A. The Km values of the three substrates and the kcat values of the six reactions are given. For aminoacylation at the terminal 2'OH group of the tRNA differences of 13.38 and 13.17 kJ in binding energies between valine and isoleucine have been calculated which result in discrimination factors of 181 and 167. For aminoacylation at the terminal 3'-OH group a difference of only 4.43 kJ and a low discrimination factor of only 6 is observed. Thus maximal discrimination between the cognate and the noncognate amino acid is only achieved in aminoacylation at the 2'-OH group and conclusions drawn from experiments with modified tRNAs concerning 2',3'-specificity have led to correct results in spite of different catalytic cycles in aminoacylation of the natural and the modified tRNAs. The stability of Ile-tRNAIle-C-C-2'dA and Val-tRNAIle-C-C-2'dA, the lesser stability of Val-tRNAVal-C-C-2'dA and the instability of Thr-tRNAVal-C-C-2'dA are consistent with postulations for a 'pre-transfer' proofreading step for isoleucyl-tRNA synthetase and a 'post-transfer' hydrolytic editing step for valyl-tRNA synthetase at the terminal 3'OH group of the tRNA.     

Isoleucyl-tRNA synthetase from bakers' yeast: multistep proofreading in discrimination between isoleucine and valine with modulated accuracy, a scheme for molecular recognition by energy dissipation

For discrimination between isoleucine and valine by isoleucyl-tRNA synthetase from yeast, a multistep sequence is established. The initial discrimination of the substrates is followed by a pretransfer and a posttransfer hydrolytic proofreading process. The overall discrimination factor D was determined from kcat and Km values observed in aminoacylation of tRNAIle-C-C-A with isoleucine and valine. From aminoacylation of the modified tRNA species tRNAIle-C-C-3'dA and tRNAIle-C-C-A (3'NH2), the initial discrimination factor I (valid for the reversible substrate binding) and the proofreading factor P1 (valid for the aminoacyl adenylate formation) could be determined. Factor I was computed from ATP consumption and D1, the overall discrimination factor for this partial reaction which can be obtained from kinetic constants, and P1 was calculated from AMP formation rates. Proofreading factor P2 (valid for aminoacyl transfer reaction) was determined from AMP formation rates observed in aminoacylation of tRNAIle-C-C-A and tRNAIle-C-C-3'dA. From the initial discrimination factor I and the AMP formation rates, discrimination factor DAMP in aminoacylation of tRNAIle-C-C-A can be calculated. These values deviate by a factor II from factor D obtained by kinetics which may be due to the fact that for acylation of tRNAIle-C-C-A an initial discrimination factor I' = III is valid. The observed overall discrimination varies up to a factor of 16 according to conditions. Under optimal conditions, 38 000 correct aminoacyl-tRNAs are produced per 1 error while the energy of 5.5 ATPs is dissipated. With the determined energetic and molecular flows for the various steps of the enzymatic reaction, a coherent picture of this new type of "far away from equilibrium enzyme" emerges.     

Isoleucyl-tRNA synthetase from Escherichia coli MRE 600: discrimination between isoleucine and valine with modulated accuracy

Discrimination between isoleucine and valine is achieved with different accuracies by isoleucyl-tRNA synthetase from E. coli MRE 600. The recognition process consists of two initial discrimination steps and a pretransfer and a posttransfer proofreading event. The overall discrimination factors D were determined from kcat and Km values observed in aminoacylation of tRNA(Ile)-C-C-A with isoleucine and valine. From aminoacylation of the modified tRNA species tRNA(Ile)-C-C-A(3'NH2) initial discrimination factors I1 and pretransfer proofreading factors II1 were calculated. Factors I1 were computed from ATP consumption and D1, the overall discrimination in aminoacylation of the modified tRNA; factors II1 were calculated as quotient of AMP formation rates. Initial discrimination factors I2 and posttransfer proofreading factors II2 were determined from AMP formation rates observed in aminoacylation of tRNA(Ile)-C-C-A. The observed overall discrimination varies up to a factor of about four according to conditions. Under standard assay conditions 72,000, under optimal conditions 144,000 correct aminoacyl-tRNAs are produced per one error while 1.1 or 1.7 ATPs are consumed. A comparison with isoleucyl-tRNA synthetase from yeast shows that both enzymes act principally with the same recognition mechanism, but the enzyme from E. coli MRE 600 exhibits higher specificity and lower energy dissipation and does not show such high variation of accuracy as observed with the enzyme from yeast.     

Interaction of pseudomonic acid A with Escherichia coli B isoleucyl-tRNA synthetase.

Sodium pseudomonate was shown to be a powerful competitive inhibitor of Escherichia coli B isoleucyl-tRNA synthetase (Ile-tRNA synthetase). The antibiotic competitively inhibits (Ki 6 nM; cf. Km 6.3 microM), with respect top isoleucine, the formation of the enzyme . Ile approximately AMP complex as measured by the pyrophosphate-exchange reaction, and has no effect on the transfer of [14C]isoleucine from the enzyme . [14C]Ile approximately AMP complex to tRNAIle. The inhibitory constant for the pyrophosphate-exchange reaction was of the same order as that determined for the inhibition of the overall aminoacylation reaction (Ki 2.5 nM; cf. Km 11.1 microM). Sodium [9'-3H]pseudomonate forms a stable complex with Ile-tRNA synthetase. Gel-filtration and gel-electrophoresis studies showed that the antibiotic is only fully released from the complex by 5 M-urea treatment or boiling in 0.1% sodium dodecyl sulphate. The molar binding ratio of sodium [9'-3H]pseudomonate to Ile-tRNA synthetase was found to be 0.85:1 by equilibrium dialysis. Aminoacylation of yeast tRNAIle by rat liver Ile-tRNA synthetase was also competitively inhibited with respect to isoleucine, Ki 20 microM (cf. Km 5.4 microM). The Km values for the rat liver and E. coli B enzymes were of the same order, but the Ki for the rat liver enzyme was 8000 times the Ki for the E. coli B enzyme. This presumably explains the low toxicity of the antibiotic in mammals.     

RNA determinants for translational editing. Mischarging a minihelix substrate by a tRNA synthetase

The fidelity of protein synthesis requires efficient discrimination of amino acid substrates by aminoacyl-tRNA synthetases. Accurate discrimination of the structurally similar amino acids, valine and isoleucine, by isoleucyl-tRNA synthetase (IleRS) results, in part, from a hydrolytic editing reaction, which prevents misactivated valine from being stably joined to tRNAIle. The editing reaction is dependent on the presence of tRNAIle, which contains discrete D-loop nucleotides that are necessary to promote editing of misactivated valine. RNA minihelices comprised of just the acceptor-TPsiC helix of tRNAIle are substrates for specific aminoacylation by IleRS. These substrates lack the aforementioned D-loop nucleotides. Because minihelices contain determinants for aminoacylation, we thought that they might also play a role in editing that has not previously been recognized. Here we show that, in contrast to tRNAIle, minihelixIle is unable to trigger the hydrolysis of misactivated valine and, in fact, is mischarged with valine. In addition, mutations in minihelixIle that enhance or suppress charging with isoleucine do the same with valine. Thus, minihelixIle contains signals for charging (by IleRS) that are independent of the amino acid and, by itself, minihelixIle provides no determinants for editing. An RNA hairpin that mimics the D-stem/loop of tRNAIle is also unable to induce the hydrolysis of misactivated valine, both by itself and in combination with minihelixIle. Thus, the native tertiary fold of tRNAIle is required to promote efficient editing. Considering that the minihelix is thought to be the more ancestral part of the tRNA structure, these results are consistent with the idea that, during the development of the genetic code, RNA determinants for editing were added after the establishment of an aminoacylation system.     

Isoleucyl-tRNA synthetase from Escherichia coli MRE 600. Different pathways of the aminoacylation reaction depending on presence of pyrophosphatase, order of substrate addition in the pyrophosphate exchange, and substrate specificity with regard to ATP analogs

The substrate specificity of isoleucyl-tRNA synthetase from Escherichia coli MRE 600 with regard to ATP analogs has been compared with the results obtained with isoleucyl-tRNA synthetase from yeast. The enzyme from E. coli is less specific, the two enzymes exhibit different topographies of their active centres. The order of substrate addition to isoleucyl-tRNA synthetase from E. coli MRE 600 has been investigated by bisubstrate kinetics, product inhibition and inhibition by substrate analogs. The inhibition studies were done in the aminoacylation and in the pyrophosphate exchange reaction, the aminoacylation was investigated in the absence and presence of inorganic pyrophosphatase. As found for isoleucyl-tRNA synthetase from yeast, the results of the pyrophosphate exchange studies indicate the possibility of formation of E . Ile-AMP . ATP complexes by random addition of one ATP and one isoleucine molecule, followed by adenylate formation, release of pyrophosphate and subsequent addition of a second molecule of ATP. For the aminoacylation in the absence of pyrophosphatase, a rapid-equilibrium random ter addition of the substrates is found whereas the enzyme from yeast exhibits a steady-state ordered ter-ter mechanism; in the presence of pyrophosphatase the mechanism is bi-uni uni-bi ping-pong similarly as observed for the yeast enzyme. A comparison of inhibition patterns obtained with N(6)-benzyladenosine 5'-triphosphate under different assay conditions (spermine or magnesium ions, addition of pyrophosphatase) indicates that even more than two pathways of the aminoacylation may exist. The catalytic cycles of the two mechanisms derived from the observed orders of substrate addition and product release include the same enzyme substrate complex (E . tRNA . Ile-AMP) for the aminoacyl transfer reaction. The kcat values, however, are considerably different: kcat of the sequential pathway is about 40% lower than kcat of the ping-pong mechanism.     

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.     

Characterization of new fluorogenic substrates for the rapid and sensitive assay of cathepsin E and cathepsin D.

Cathepsin E and cathepsin D are two major intracellular aspartic proteinases implicated in the physiological and pathological degradation of intra- and extracellular proteins. In this study, we designed and constructed highly sensitive synthetic decapeptide substrates for assays of cathepsins E and D based on the known sequence specificities of their cleavage sites. These substrates contain a highly fluorescent (7-methoxycoumarin-4-yl)acetyl (MOCAc) moiety and a quenching 2,4-dinitrophenyl (Dnp) group. When the Phe-Phe bond is cleaved, the fluorescence at an excitation wavelength of 328 nm and emission wavelength of 393 increases due to diminished quenching resulting from the separation of the fluorescent and quenching moieties. The first substrate, MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Le u-Lys(Dnp)gamma-NH2, in which the Lys-Pro combination at positions P5 and P4 was designed for specific interaction with cathepsin E, is hydrolyzed equally well by cathepsins E and D (kcat/Km = 10.9 microM(-1) x s(-1) for cathepsin E and 15.6 microM(-1) x s(-1) for cathepsin D). A very acidic pH optimum o was obtained for both enzymes. The second substrate, MOCAc-Gly-Lys-Pro-Ile-Ile-Phe-Phe-Arg-Le u-Lys(Dnp)gamma-NH2, in which the isoleucine residue at position P2 was meant to increase the specificity for cathepsin E, is also hydrolyzed equally by both enzymes (kcat/Km = 12.2 microM(-1) x s(-1) for cathepsin E and 16.3 microM(-1) x s(-1) for cathepsin D). The kcat/Km values for both substrates are greater than those for the best substrates for cathepsins E and D described so far. Unfortunately, each substrate shows little discrimination between cathepsin E and cathepsin D, suggesting that amino acids at positions far from the cleavage site are important for discrimination between the two enzymes. However, in combination with aspartic proteinase inhibitors, such as pepstatin A and Ascaris pepsin inhibitor, these substrates enable a rapid and sensitive determination of the precise levels of cathepsins E and D in crude cell extracts of various tissues and cells. Thus these substrates represent a potentially valuable tool for routine assays and for mechanistic studies on cathepsins E and D.     

The amino-acid residues on the C-terminal side of the cleavage site of angiotensinogen influence the species specificity of reaction with renin

The N-terminal sequences of human and canine angiotensinogen and two hybrid sequences were synthesized and used to determine whether the species specificity of renin is influenced by amino-acid residues adjacent to the cleavage site. kcat/Km for the generation of angiotensin I from the N-terminal tridecapeptide of human angiotensinogen by canine renin is 0.37% of that observed when the N-terminal tetradecapeptide from canine angiotensinogen is used as a substrate. Replacement of the valine residue at P'1 in the human tridecapeptide with the leucine residue from the canine sequence triples kcat and improves Km 4-fold. Replacement of isoleucine residue at P'2 with the valine residue from the canine sequence enhances Km 8-fold. Substitution of the histidine residue at P'3 with the tyrosine serine sequence of canine angiotensinogen increases kcat an order of magnitude. Results obtained with the synthetic substrate are similar to those observed with the protein substrates. Canine renin does not cleave human angiotensinogen. Also, kcat/Km of canine renin for its homologous substrate is about 6-times greater than the kcat/Km value for human renin acting on human angiotensinogen.     

Lysyl-tRNA synthetase from yeast. Discrimination of amino acids by native and phosphorylated species.

Discrimination factors (D) which are characteristic for discrimination between lysine and 19 naturally occurring non-cognate amino acids have been determined from kcat and Km values for native and phosphorylated lysyl-tRNA synthetases from yeast. Generally, both species of this class II aminoacyl-tRNA synthetase are considerably less specific than the class I synthetases specific for isoleucine, valine, tyrosine, and arginine. D values of the native enzyme are in the range 90-1700, D values of the phosphorylated species in the range 40-770. The phosphorylated enzyme acts faster and less accurately. In aminoacylation of tRNALys-C-C-A(2'NH2) discrimination factors D1 vary over 30-980 for the native and over 8-300 for the phosphorylated enzyme. From AMP formation stoichiometry and D1 values pretransfer proof-reading factors (II1) of 1.1-56 were calculated for for the native enzyme, factors of 1.0-44 for the phosphorylated species. Post-transfer proof-reading factors (II2) were calculated from D values and AMP formation stoichiometry in acylation of tRNALys-C-C-A. Pretransfer proof-reading is the main correction step, posttransfer proof-reading is less effective or negligible (II2 approximately 1-8). Initial discrimination factors (I), which are due to differences in Gibbs free energies of binding between lysine and noncognate substrates (delta delta GI), were calculated from discrimination and proof-reading factors. In contrast to class I synthetases, for lysyl-tRNA synthetase only one initial discrimination step can be assumed and amino acid recognition is reduced to a three-step process instead of the four-step recognition observed for the class I synthetases. Plots of delta delta GI values against accessible surface areas of amino acids show clearly that phosphorylation of the enzyme changes the structures of the amino acid binding sites. This is illustrated by a hypothetical 'stopper model' of these sites.     

Kinetic Proofreading at Single Molecular Level: Aminoacylation of tRNAIle and the Role of Water as an Editor

Proofreading/editing in protein synthesis is essential for accurate translation of information from the genetic code. In this article we present a theoretical investigation of efficiency of a kinetic proofreading mechanism that employs hydrolysis of the wrong substrate as the discriminatory step in enzyme catalytic reactions. We consider aminoacylation of tRNA^Ile which is a crucial step in protein synthesis and for which experimental results are now available. We present an augmented kinetic scheme and then employ methods of stochastic simulation algorithm to obtain time dependent concentrations of different substances involved in the reaction and their rates of formation. We obtain the rates of product formation and ATP hydrolysis for both correct and wrong substrates (isoleucine and valine in our case, respectively), in single molecular enzyme as well as ensemble enzyme kinetics. The present theoretical scheme correctly reproduces (i) the amplitude of the discrimination factor in the overall rates between isoleucine and valine which is obtained as (1.8×10²).(4.33×10²) = 7.8×10⁴, (ii) the rates of ATP hydrolysis for both Ile and Val at different substrate concentrations in the aminoacylation of tRNA^Ile. The present study shows a non-michaelis type dependence of rate of reaction on tRNA^Ile concentration in case of valine. The overall editing in steady state is found to be independent of amino acid concentration. Interestingly, the computed ATP hydrolysis rate for valine at high substrate concentration is same as the rate of formation of Ile-tRNA^Ile whereas at intermediate substrate concentration the ATP hydrolysis rate is relatively low. We find that the presence of additional editing domain in class I editing enzyme makes the kinetic proofreading more efficient through enhanced hydrolysis of wrong product at the editing CP1 domain.     

The mechanism of ATP inhibition of wild type and mutant phosphofructo-1-kinase from Escherichia coli.

Escherichia coli 6-phosphofructo-1-kinase was inhibited by high concentrations of ATP at alkaline pH. The mechanism of the inhibition was studied with two mutants generated by site-directed mutagenesis; I126A, with a Km for fructose-6-P that was more than two orders of magnitude higher than that of wild type but with minimal changes in kcat and Km for ATP, and R72H, with little change in substrate half-saturation concentrations but with a kcat that was 300-fold lower that of wild type enzyme. ATP and fructose-6-P interacted in a mutually antagonistic manner; that is ATP decreased the apparent affinity for fructose-6-P and vice versa. The half-saturation concentrations for both substrates, most strikingly fructose-6-P, increased with increasing pH while the kcat increased. Studies with I126A suggested that ATP inhibition was not dependent on a dissociable group with a pK in the alkaline range and that the inhibition was not caused by abortive binding of substrate to the wrong substrate site. Inhibition was not the result of differential affinity of ATP for the R and T states of the enzyme. The low kcat mutant, R72H, did not display ATP inhibition. These data indicate that ATP inhibition results from substrate antagonism coupled with a steady state random mechanism wherein the high rate of catalysis does not permit equilibration of substrates.     

Use of a fluorescence plate reader for measuring kinetic parameters with inner filter effect correction

A general method is presented here for the determination of the Km, kcat, and kcat/Km of fluorescence resonance energy transfer (FRET) substrates using a fluorescence plate reader. A simple empirical method for correcting for the inner filter effect is shown to enable accurate and undistorted measurements of these very important kinetic parameters. Inner filter effect corrected rates of hydrolysis of a FRET peptide substrate by hepatitis C virus (HCV) NS3 protease at various substrate concentrations enabled measurement of a Km value of 4.4 +/- 0.3 microM and kcat/Km value of 96,500 +/- 5800 M-1 s-1. These values are very close to the HPLC-determined Km value of 4.6 +/- 0.7 microM and kcat/Km value of 92,600 +/- 14,000 M-1 s-1. We demonstrate that the inner filter effect correction of microtiter plate reader velocities enables rapid measurement of Ki and Ki' values and kinetic inhibition mechanisms for HCV NS3 protease inhibitors.     

Mammalian protein methylesterase. Physical and enzymic properties.

Protein methylesterase (PME) amino acid composition and substrate specificity towards methylated normal and deamidated protein substrates were investigated. The enzyme contained 23% acidic and 5% basic residues. These values are consistent with a pI of 4.45. The product formed from methylated protein by PME was confirmed as methanol by h.p.l.c. The kcat. and Km values for several methylated protein substrates ranged from 20 x 10(-6) to 560 x 10(-6) s-1 and from 0.5 to 64 microM respectively. However, the kcat./Km ratios ranged within one order of magnitude from 11 to 52 M-1.s-1. Results with the irreversible cysteine-proteinase inhibitor E-64 suggested that these low values were in part due to the fact that only one out of 25 molecules in the PME preparations was enzymically active. When PME was incubated with methylated normal and deamidated calmodulin, the enzyme hydrolysed the latter substrate at a higher rate. The Km and kcat. for methylated normal calmodulin were 0.9 microM and 31 x 10(-6) s-1, whereas for methylated deamidated calmodulin values of 1.6 microM and 188 x 10(-6) s-1 were obtained. The kcat./Km ratios for methylated normal and deamidated calmodulin were 34 and 118 M-1.s-1 respectively. By contrast, results with methylated adrenocorticotropic hormone (ACTH) substrates indicated that the main difference between native and deamidated substrates resides in the Km rather than the kcat. The Km for methylated deamidated ACTH was 5-fold lower than that for methylated native ACTH. The kcat./Km ratios for methylated normal and deamidated ACTH were 43 and 185 M-1.s-1 respectively. These results indicate that PME recognizes native and deamidated methylated substrates as two different entities. This suggests that the methyl groups on native calmodulin and ACTH substrates may not be on the same amino acid residues as those on deamidated calmodulin and ACTH substrates.     

Reverse reaction of smooth muscle myosin light chain kinase. Formation of ATP from phosphorylated light chain plus ADP

Incubation of smooth muscle phosphorylated heavy meromyosin in the presence of myosin light chain kinase, calmodulin, ADP, and Ca2+ results in a decrease of the protein-bound phosphate. The dephosphorylation is not due to phosphatase activity and is dependent on the presence of ADP and the active ternary myosin light chain kinase complex. Using 32P-labeled phosphorylated 20,000-dalton light chains as the phosphate donor, the formation of ATP from ADP can be demonstrated. This reaction requires the presence of Ca2+, calmodulin, and myosin light chain kinase. These results indicate that myosin light chain kinase can catalyze a reverse reaction and form ATP from ADP and phosphorylated substrate. The rate of the reverse reaction, kcat/KLC approximately 0.21 min-1 microM-1, is considerably slower than the forward reaction under similar conditions and is therefore detectable only at relatively high concentrations of myosin light chain kinase. For the reverse reaction, KmADP is approximately 30 microM and ATP is a competitive inhibitor, KIATP approximately 88 microM. For the forward reaction, measured with both isolated light chains and intact myosin, KmATP is approximately 100 microM and ADP is a competitive inhibitor, KiADP approximately 140 microM (myosin) and 120 microM (light chains). Thus, the affinity of ATP for the forward and reverse reactions is similar, but the affinity of ADP is higher for the reverse reaction. From the light chain dependence of the two reactions, the following was calculated: forward, Km = 5 microM, kcat = 1720 min-1, and reverse, Km = 130 microM, kcat = 27 min-1. In contrast to the data obtained with isolated light chains, it is suggested that, with intact myosin as substrate, the Km term is primarily responsible for determining the rate of the reverse reaction. With light chains phosphorylated at serine 19 and threonine 18, it was shown that both sites act as a phosphate donor, although the reverse reaction for threonine 18 is slower than that for serine 19.     

ilvU, a locus in Escherichia coli affecting the derepression of isoleucyl-tRNA synthetase and the RPC-5 chromatographic profiles of tRNAIle and tRNAVal.

A mutation in the ilvU locus of Escherichia coli has led to a complex phenotype that included resistance to thiaisoleucine, a loss of derepressibility of isoleucyl tRNA synthetase, and an alteration of the RPC-5 chromatographic profile of the branched-chain aminoacyl-tRNA's. The alterations were manifest in an increase in the amount of Species 2 of both tRNAIle and tRNAVal at the expense of Species 1. A similar alteration, but independent of (and additive to) that caused by the ilvU mutation, was observed upon limitation of either isoleucine or valine. The shift in profile caused by limitation was also independent of the reduced growth rate or the derepression of the isoleucine and valine biosynthetic enzymes that also result from limitation. During chloramphenicol treatment nearly all tRNAIle and tRNAVal formed appears as species 2. Upon recovery from chloramphenicol, Species 2 of both acceptors are converted to Species 1. It is proposed that the ilvU product not only allows derepression of isoleucyl-tRNA synthetase but also retards the conversion of tRNA2Ile to tRNA1Ile and that of tRNA2Val to tRNA1Val. The mutated ilvU loci abolish the derepression and are more efficient in retarding the conversion.     

Characterization of 2-ketoisovalerate ferredoxin oxidoreductase, a new and reversible coenzyme A-dependent enzyme involved in peptide fermentation by hyperthermophilic archaea.

Cell extracts of the proteolytic and hyperthermophilic archaea Thermococcus litoralis, Thermococcus sp. strain ES-1, Pyrococcus furiosus, and Pyrococcus sp. strain ES-4 contain an enzyme which catalyzes the coenzyme A-dependent oxidation of branched-chain 2-ketoacids coupled to the reduction of viologen dyes or ferredoxin. This enzyme, termed VOR (for keto-valine-ferredoxin oxidoreductase), has been purified from all four organisms. All four VORs comprise four different subunits and show amino-terminal sequence homology. T. litoralis VOR has an M(r) of ca. 230,000, with subunit M(r) values of 47,000 (alpha), 34,000 (beta), 23,000 (gamma), and 13,000 (delta). It contains about 11 iron and 12 acid-labile sulfide atoms and 13 cysteine residues per heterotetramer (alpha beta gamma delta), but thiamine pyrophosphate, which is required for catalytic activity, was lost during purification. The most efficient substrates (kcat/Km > 1.0 microM-1 s-1; Km < 100 microM) for the enzyme were the 2-ketoacid derivatives of valine, leucine, isoleucine, and methionine, while pyruvate and aryl pyruvates were very poor substrates (kcat/Km < 0.2 microM-1 s-1) and 2-ketoglutarate was not utilized. T. litoralis VOR also functioned as a 2-ketoisovalerate synthase at 85 degrees C, producing 2-ketoisovalerate and coenzyme A from isobutyryl-coenzyme A (apparent Km, 250 microM) and CO2 (apparent Km, 48 mM) with reduced viologen as the electron donor. The rate of 2-ketoisovalerate synthesis was about 5% of the rate of 2-ketoisovalerate oxidation. The optimum pH for both reactions was 7.0. A mechanism for 2-ketoisovalerate oxidation based on data from substrate-induced electron paramagnetic resonance spectra is proposed, and the physiological role of VOR is discussed.     

Substrate recognition by the hetero-octameric ATP phosphoribosyltransferase from Lactococcus lactis

Two families of ATP phosphoribosyl transferases (ATP-PRT) join ATP and 5-phosphoribosyl-1 pyrophosphate (PRPP) in the first reaction of histidine biosynthesis. These consist of a homohexameric form found in all three kingdoms and a hetero-octameric form largely restricted to bacteria. Hetero-octameric ATP-PRTs consist of four HisGS catalytic subunits related to periplasmic binding proteins and four HisZ regulatory subunits that resemble histidyl-tRNA synthetases. To clarify the relationship between the two families of ATP-PRTs and among phosphoribosyltransferases in general, we determined the steady state kinetics for the hetero-octameric form and characterized the active site by mutagenesis. The KmPRPP (18.4 +/- 3.5 microM) and kcat (2.7 +/- 0.3 s-1) values for the PRPP substrate are similar to those of hexameric ATP-PRTs, but the Km for ATP (2.7 +/- 0.3 mM) is 4-fold higher, suggestive of tighter regulation by energy charge. Histidine and AMP were determined to be noncompetitive (Ki = 81.1 microM) and competitive (Ki = 1.44 mM) inhibitors, respectively, with values that approximate their intracellular concentrations. Mutagenesis experiments aimed at investigating the side chains recognizing PRPP showed that 5'-phosphate contacts (T159A and T162A) had the largest (25- and 155-fold, respectively) decreases in kcat/Km, while smaller decreases were seen with mutants making cross subunit contacts (K50A and K8A) to the pyrophosphate moiety or contacts to the 2'-OH group. Despite their markedly different quaternary structures, hexameric and hetero-octameric ATRP-PRTs exhibit similar functional parameters and employ mechanistic strategies reminiscent of the broader PRT superfamily.     

Mechanistic studies on thrombin catalysis

The kinetic mechanism of the cleavage of four p-nitroanilide (pNA) substrates by human alpha-thrombin has been investigated by using a number of steady-state kinetic techniques. Solvent isotope and viscosity effects were used to determine the stickiness of the substrates at the pH optimum of the reaction; a sticky substrate is defined as one that undergoes catalysis faster than it dissociates from the Michaelis complex. Whereas benzoyl-Arg-pNA could be classified as a nonsticky substrate, D-Phe-pipecolyl-Arg-pNA was very sticky. The other two substrates (tosyl-Gly-Pro-Arg-pNA and acetyl-D-Phe-pipecolyl-Arg-pNA) were slightly sticky. The pH profiles of kcat/Km were bell-shaped for all substrates. The pKa values determined from the pH dependence of kcat/Km for benzoyl-Arg-pNA were about 7.5 and 9.1. Similar pKa values were determined from the pH profiles of kcat/Km for tosyl-Gly-Pro-Arg-pNA and acetyl-D-Phe-pipecolyl-Arg-pNA and for the binding of the competitive inhibitor N alpha-dansyl-L-arginine-4-methylpiperidine amide. The groups responsible for the observed pKa values were proposed to be His57 and the alpha-amino group of Ile16. The temperature dependence of the pKa values was consistent with this assignment. The pKa values of 6.7 and 8.6 observed in the pH profile of kcat/Km for D-Phe-pipecolyl-Arg-pNA were displaced to lower values than those observed for the other substrates. The displacement of the acidic pKa value could be attributed to the stickiness of this substrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Active transport of nonpolar amino acids in Chromatium vinosum

The photosynthetic purple sulfur bacterium, Chromatium vinosum, takes up the amino acids, L-phenylalanine and L-leucine, via two apparently different electrogenic, H+/amino acid symports. Na+ serves as an allosteric modulator for leucine transport, lowering the Km for leucine from 66 to 15 microM. C. vinosum cells also contain a system that transports both isoleucine and valine. The isoleucine/valine system has the attributes of a H+/amino acid symport at pH less than 7.5 but appears to function as a H+/Na+ (Li+)/amino acid symport at pH greater than or equal to 7.5. Na+ gradients produce an allosteric lowering of the Km values for both isoleucine and valine, from 14 to 7 microM and from 34 to 17 microM, respectively. C. vinosum also accumulates D-alanine in an energy-dependent reaction. The transport process appears to involve the electrogenic cotransport of D-alanine and Na+. The Km value for D-alanine was determined to be 9 microM. Unlike the previously characterized C. vinosum L-alanine/Na+ symport, Na+ gradients did not affect the Km for D-alanine transport. L-Alanine and glycine, but not alpha-aminoisobutyric acid, act as competitive inhibitors for D-alanine transport.