The plant aminoacyl-tRNA synthetases. Effect of sodium chloride on tRNA aminoacylation and aminoacyl-tRNA decomposition catalysed by aminoacyl-tRNA synthetases from yellow lupin seeds.

The initial velocity and the extent of aminoacylation are affected by sodium chloride in the lupin aminoacylation systems involving serine, isoleucine, lysine, leucine, phenylalanine and valine. Pyrophosphorolysis and enzymatic hydrolysis of [14C]Val-tRNA catalysed by lupin valyl-tRNA synthetase are inhibited by sodium chloride nearly to the same extent. Evidence is presented that when a limiting amount of synthetase is used, the equilibrium of the aminoacylation reaction in the lupin valine system is determined only by the rate of aminoacylation and non-enzymatic deacylation of aminoacyl-tRNA, the former but not the latter reaction being dependent on concentration of the enzyme and monovalent salt.     

An in vivo-in vitro alkaline DNA unwinding assay for hepatic DNA damage: comparison with the alkaline sucrose gradient centrifugation technique

The addition of glycerol, sucrose, or other diol-containing reagents to solutions of aminoacyl-tRNA (aa-tRNA) substantially increased the rate of hydrolysis of the aminoacyl ester bond. Glycerol at 4.9% (v/v) doubled the rate of deacylation for several aa-tRNAs and peptidyl-tRNAs, including fMet-tRNAMetf, while 1% (v/v) glycerol increased the deacylation rate by 20%. This effect was not caused by a nuclease contamination, and tRNA deacylated in the presence of glycerol could be fully recharged. The deacylation of aa-tRNA was accelerated by glycerol and sucrose even in the presence of EF-Tu X GTP. In addition, the extent of tRNA aminoacylation was reduced when glycerol was present at concentrations above 2% (v/v). Thus, glycerol and sucrose are not necessarily inert or neutral additions to an in vitro incubation.     

Characteristics of thermal inactivation of lysozyme in solution

In the buffer solution (pH 6,2) at 20-80 degrees, the lysozyme thermoinactivation was studied by monitoring of its activity decrease in the lysis of M. lysodeicticus cells. Protein inactivation was characterized by effective pseudofirst order rate constants which depend on enzyme concentration and are described by equation k = k0 . exp [-alpha 0 (1-gamma/T) [E]0], where k0 is inactivation rate constant at "infinite" enzyme dilution, [E0] is an initial lysozyme concentration, alpha 0 and gamma are the coefficients independent on [E0]. By extrapolation of the "k" dependencies on [E]0 the constants k0 were determined. In the range 40-70 degrees C, the rate constant k0 is equal 4,0 X 10(11) . exp (-24 200/RT) sec-1.     

The C-terminal domain of the archaeal leucyl-tRNA synthetase prevents misediting of isoleucyl-tRNA(Ile)

In the archaeal leucyl-tRNA synthetase (LeuRS), the C-terminal domain recognizes the long variable arm of tRNA(Leu) for aminoacylation, and the so-called editing domain deacylates incorrectly formed Ile-tRNA(Leu). We previously reported, for Pyrococcus horikoshii LeuRS, that a deletion mutant lacking the C-terminal domain (LeuRS_delta(811-967)) retains normal editing activity, but has severely reduced aminoacylation activity. In this study, we found that LeuRS_delta(811-967), but not the wild-type LeuRS, exhibited surprisingly robust deacylation activity against Ile-tRNA(Ile), correctly formed by isoleucyl-tRNA synthetase ("misediting"). Structural superposition of tRNA(Ile) onto the LeuRS x tRNA(Leu) complex indicated that Ile911, Lys912, and Glu913 of the LeuRS C-terminal domain clash with U20 of tRNA(Ile), which is bulged out as compared to the corresponding nucleotide of tRNA(Leu). The deletion of amino acid residues 911-913 of LeuRS enhanced the Ile-tRNA(Ile) deacylation activity, without affecting the Ile-tRNA(Leu) deacylation activity. These results demonstrate that the clashing between U20 of tRNA(Ile) and residues 911-913 of the LeuRS C-terminal domain is the structural mechanism that prevents misediting. In contrast, the deletion of the C-terminal domains of the isoleucyl- and valyl-tRNA synthetases impaired both the aminoacylation (Ile-tRNA(Ile) and Val-tRNA(Val) formation, respectively) and editing (Val-tRNA(Ile) and Thr-tRNA(Val) deacylation, respectively) activities, and did not cause misediting (Val-tRNA(Val) and Thr-tRNA(Thr) deacylation, respectively) activity. Thus, the requirement of the C-terminal domain for misediting prevention is unique to LeuRS, which does not recognize the anticodon of the cognate tRNA, unlike the common aminoacyl-tRNA synthetases.     

Investigation of the interactions of oxytocin with neurophysins at low pH using carbon-13 nuclear magnetic resonance and carbon-13-labeled hormones.

The specifically 13C-labeled (90% 13C-enriched) peptide hormone derivatives [1-hem[2-13C]cystine]oxytocin, [1-hemi[1-13C]cystine]oxytocin, and [2-[-2-13C]tyrosine[-oxytocin and the analogue [3-[2-13C]leucine]oxytocin were prepared by total synthesis and used to study the interactions of the neurohypophyseal hormones with the bovine neurophysins as a function of pH and temperature. Under all conditions, whether high or low pH, the chemical shifts of the labeled carbon atoms of the bound hormones are the same, but they are shifted significantly from their positions in the free hormone. These results indicate that interactions of the side chain and disulfide moieties of the hormone with the neurophysins do not change as a function of pH. At neutral pH and 20--35 degrees C, the labeled atoms of the hormone are in slow exchange (1--5 s-1) with the neurophysins for the above hormone derivatives, but at low pH they are in intermediate or fast exchange depending upon the pH and temperature. At low pH, the dissociation rate constant (koff) is about 100-fold greater than the value at neutral pH, and this increase appears to be due exclusively to the breaking of the salt bridge involving the N-terminal amino group of oxytocin and a side-chain carboxyl group of neurophysin. Since the dissociation constant (Kd) also increases by about 100-fold in going from neutral to low pH, the association rate constant is deduced to be the same at neutral and low pH. In contrast to the low pH results, an increase in pH (from 6.6 to 10.5) leads to a continual decrease in the binding constant but to no apparent change in the dissociation rate constant. The bound hormone is always in slow exchange at high pH, even when the binding constant has been reduced by 2 or 3 orders of magnitude. At high pH, the decrease in binding affinity is due solely to the deprotonation of the alpha-amino group of the free hormone. Thus, at high pH the apparent association rate constant decreases, while the dissociation rate constant remains unchanged.     

Role of glutamate-268 in the catalytic mechanism of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans

Nonphosphorylating nicotinamide adenine dinucleotide (phosphate)- [NAD(P)-] dependent aldehyde dehydrogenases share a number of conserved amino acid residues, several of which are directly implicated in catalysis. In the present study, the role of Glu-268 from nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Streptococcus mutans was investigated. Its substitution by Ala resulted in a k(cat) decrease by 3 orders of magnitude. Pre-steady-state analysis showed that, for both the wild-type and E268A GAPNs, the rate-limiting step of the reaction is associated with deacylation. The pH dependence of the rate of acylation of wild-type GAPN is characterized by the contributions of distinct enzyme protonic species with two pK(a)s of 6.2 and 7.5. Substitution of Glu-268 by Ala resulted in a monosigmoidal pH dependence of the rate constant of acylation with a pK(a) of 6.2, which suggested the assignment of pK(a) 7.5 to Glu-268. Moreover, the E268A substitution did not significantly affect the efficiency of acylation of GAPN, showing that Glu-268 is not critically involved in the acylation, which includes Cys-302 nucleophilic activation and hydride transfer. On the contrary, the drastic decrease of the steady-state rate constant for the E268A GAPN demonstrated the essential role of Glu-268 in the deacylation. At basic pH, the solvent isotope effect of 2.3, characterized by a unique pK(a) of 7.7, and the linearity of the proton inventory showed that the rate-limiting process for deacylation is associated with the hydrolysis step and suggested that the glutamate form of Glu-268 acts as a base catalyst in this process. Surprisingly, the double-sigmoidal form of the pH-steady-state rate constant profile, characterized by pK(a) values of 6.1 and 7.4, revealed the high efficiency of the deacylation even at pH lower than 7.4. Therefore, we propose that the major role of Glu-268 is to promote deacylation through activation and orientation of the attacking water molecule, and in addition to act as a base catalyst at basic pH. From these results in relation to those recently described [Marchal, S., and Branlant, G. (1999) Biochemistry 38, 12950-12958], a scenario for the chemical catalysis of GAPN is proposed.     

Changes in pH associated with clotting of fibrinogen. Kinetic studies of the pH shift and correlation of the pH change with the release of fibrinopeptides and the ensuing polymerization

The effect of the initial pH and the concentrations of thrombin, fibrinogen, and Ca2+ upon the rate of pH change associated with clotting of bovine fibrinogen by human thrombin was investigated at pH 6.80, 7.80, and 8.80, 0.3 ionic strength, 25 degrees C, and 19.5 mg/mL final fibrinogen concentration. At pH 6.80 and 7.80, the reaction was first order, with rate constant k1. At pH 8.80, a first-order reaction of the release of H+ (k1) was followed by a partial rebinding of these in a reaction consecutive to the first one (k2). At each of the above pH values, k1 was proportional to thrombin concentration in the 0.05-3.0 min-1 range investigated. The k1 constants were 0.111 +/- 0.001, 0.250 +/- 0.005, and 0.190 +/- 0.002 min-1 (NIH thrombin units)-1 mL-1 at pH 6.80, 7.80, and 8.80, respectively. Plots of log rate vs log thrombin concentration of these data were linear with slopes close to 1 at all three pH values. The rate of the second reaction (k2) was independent of both the thrombin and the initial fibrinogen concentration. The pH dependence of k1 exhibited a bell-shaped curve that could be resolved into the effect of one group with a pK of 7.27 that increased the rate and another with a pK of 9.22 that decreased the rate. With constant thrombin concentration but varying fibrinogen concentration, plots of 1/k1 vs [fibrinogen] were linear, but the lines did not pass through the origin. From the slope and intercept, kcat and KM of the Michaelis-Menten equation could be calculated. The same parameters were obtained also from initial velocity vs [fibrinogen] plots. Values of kcat were consistent and accurate; those of KM were more scattered. KM was (22.4-34.2) X 10(-6) M at pH 6.80 and approximately 7 X 10(-6) M in the pH 7.26-8.80 range. The latter value, pertaining to the release of H+ ions, is in agreement with values in the literature for KM of the release of fibrinopeptide A by thrombin in the 7.4-8.0 pH range. The value of kcat s-1 (unit of thrombin)-1 mL-1 increases from 1.2 X 10(-10) s-1 unit of thrombin-1 mL-1 at pH 6.80 to 2.46 X 10(-10) at pH 7.80 and then decreases to 2.01 X 10(-10) 10(-1) (units of thrombin)-1 mL-1 at pH 8.80. The kcat values are significantly lower than those in the literature for the release of fibrinopeptide A.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis and characterization of a new fluorogenic active-site titrant of serine proteases

The molecule 3',6'-bis(4-guanidinobenzoyloxy)-5-[N'-(4-carboxyphenyl)thioureido[spirop]isobenzofuran-1-(3H),9'-[9H]xanthen]-3-one, abbreviated FDE, was designed and synthesized as a fluorogenic active-site titrant for serine proteases. It is an analogue of p-nitrophenyl p-guanidino-benzoate (NPGB) in which a fluorescein derivative is substituted for p-nitrophenol. FDE and NPGB exhibit similar kinetic characteristics in an active-site titration of trypsin in phosphate-buffered saline, pH 7.2. The rate of acylation with FDE is extremely fast (k2 = 1.05 s-1) and the rate of deacylation extremely slow (k3 = 1.66 X 10(-5) s-1). The Ks is 3.06 X 10(-6) M, and the Km(app) is 4.85 X 10(-11) M. With two of the serine proteases involved in fibrinolysis, the rate of acylation with FDE is also fast, K2 = 0.112 s-1 for urokinase and 0.799 s-1 for plasmin, and the rate of deacylation is slow, k3 = 3.64 X 10(-4) s-1 for urokinase and 6.27 X 10(-6) s-1 for plasmin. The solubility limit of FDE in phosphate-buffered saline is 1.3 X 10(-5) M, and the first-order rate constant for spontaneous hydrolysis is 5.1 X 10(-6) s-1. The major difference between FDE and NPGB is the detectability of the product in an active-site titration. p-Nitrophenol can be detected at concentrations no lower than 10(-6) M whereas fluorescein can be detected at concentrations as low as 10(-12) M. Thus, FDE should be useful in quantitatively assaying serine proteases as very low concentrations.     

Role of intramitochondrial pH in the energetics and regulation of mitochondrial oxidative phosphorylation.

The dependence of ATP synthesis coupled to electron transfer from 3-hydroxy-butyrate (3-OH-B) to cytochrome c on the intramitochondrial pH (pHi) was investigated. Suspensions of isolated rat liver mitochondria were incubated at constant extramitochondrial pH (pHe) with ATP, ADP, Pi, 3-OH-B, and acetoacetate (acac) (the last two were varied to maintain [3-OH-B]/[acac] constant), with or without sodium propionate to change the intramitochondrial pH. Measurements were made of the steady-state water volume of the mitochondrial matrix, transmembrane pH difference, level of cytochrome c reduction, concentration of metabolites and rate of oxygen consumption. For each experiment, conditions were used for which transmembrane pH was near maximal and minimal values and the measured extramitochondrial [ATP], [ADP], and [Pi] were used to calculate log[ATP]/[ADP][Pi]. When [3-OH-B]/[acac] and [cyt c2+]/[cyt c3+] were constant, and pHi was decreased from approx. 7.7 to 7.2, log [ATP]/[ADP][Pi] at high pHi was significantly (P less than 0.02) greater than at low pHi. The mean slope (delta log [ATP]/[ADP][Pi] divided by the change in pHi) was 1.08 +/- 0.15 (mean +/- S.E.). This agrees with the slope of 1.0 predicted if the energy available for ATP synthesis is dependent upon the pH at which 3-hydroxybutyrate dehydrogenase operates, that is, on the pH of the matrix space. The steady-state respiratory rate and reduction of cytochrome c were measured at different pHi and pHe values. Plots of respiratory rate vs.% cytochrome c reduction at different intra- and extramitochondrial pH values indicated that the respiratory rate is dependent upon pHi and not on pHe. This implies that the matrix space is the source of protons involved in the reduction of oxygen to water in coupled mitochondria.     

F-actin sequesters elongation factor 1alpha from interaction with aminoacyl-tRNA in a pH-dependent reaction

The machinery of eukaryotic protein synthesis is found in association with the actin cytoskeleton. A major component of this translational apparatus, which is involved in the shuttling of aa-tRNA, is the actin- binding protein elongation factor 1alpha (EF-1alpha). To investigate the consequences for translation of the interaction of EF-1alpha with F- actin, we have studied the effect of F-actin on the ability of EF- 1alpha to bind to aa-tRNA. We demonstrate that binding of EF-1alpha:GTP to aa-tRNA is not pH sensitive with a constant binding affinity of approximately 0.2 microM over the physiological range of pH. However, the sharp pH dependence of binding of EF-1alpha to F-actin is sufficient to shift the binding of EF-1alpha from F-actin to aa-tRNA as pH increases. The ability of EF-1alpha to bind either F-actin or aa- tRNA in competition binding experiments is also consistent with the observation that EF-1alpha's binding to F-actin and aa-tRNA is mutually exclusive. Two pH-sensitive actin-binding sequences in EF-1alpha are identified and are predicted to overlap with the aa-tRNA-binding sites. Our results suggest that pH-regulated recruitment and release of EF- 1alpha from actin filaments in vivo will supply a high local concentration of EF-1alpha to facilitate polypeptide elongation by the F-actin-associated translational apparatus.     

The pH dependence of serpin-proteinase complex dissociation reveals a mechanism of complex stabilization involving inactive and active conformational states of the proteinase which are perturbable by calcium

Serpin family protein proteinase inhibitors trap proteinases at the acyl-intermediate stage of cleavage of the serpin as a proteinase substrate by undergoing a dramatic conformational change, which is thought to distort the proteinase active site and slow deacylation. To investigate the extent to which proteinase catalytic function is defective in the serpin-proteinase complex, we compared the pH dependence of dissociation of several serpin-proteinase acyl-complexes with that of normal guanidinobenzoyl-proteinase acyl-intermediate complexes. Whereas the apparent rate constant for dissociation of guanidinobenzoyl-proteinase complexes (k(diss, app)) showed a pH dependence characteristic of His-57 catalysis of complex deacylation, the pH dependence of k(diss, app) for the serpin-proteinase complexes showed no evidence for His-57 involvement in complex deacylation and was instead characteristic of a hydroxide-mediated deacylation similar to that observed for the hydrolysis of tosylarginine methyl ester. Hydroxylamine enhanced the rate of serpin-proteinase complex dissociation but with a rate constant for nucleophilic attack on the acyl bond several orders of magnitude slower than that of hydroxide, implying limited accessibility of the acyl bond in the complex. The addition of 10-100 mm Ca(2+) ions stimulated up to 80-fold the dissociation rate constant of several serpin-trypsin complexes in a saturable manner at neutral pH and altered the pH dependence to a pattern characteristic of His-57-catalyzed complex deacylation. These results support a mechanism of kinetic stabilization of serpin-proteinase complexes wherein the complex is trapped as an acyl-intermediate by a serpin conformational change-induced inactivation of the proteinase catalytic function, but suggest that the inactive proteinase conformation in the complex is in equilibrium with an active proteinase conformation that can be stabilized by the preferential binding of an allosteric ligand such as Ca(2+).     

Stimulation of valyl- and isoleucyl-tRNA synthetase reactions by polyamines

The aminoacylation of tRNA catalysed by valyl-tRNA synthetase (EC 6.1.1.9) and isoleucyl-tRNA synthetase (EC 6.1.1.5) fromMycobacterium smegmatis is dependent on the presence of divalent metal ions. Polyamines alone, in the absence of metal ions, do not bring about aminoacylation. In the presence of suboptimal concentrations of Mg²⁺, polyamines significantly stimulate the reaction. Of the cations tested, only Mn²⁺, Co²⁺ and Ca²⁺ can partially substitute for Mg²⁺ in aminoacylation, and spermine stimulates aminoacylation in the presence of these cations also. At neutral pH, spermine deacylates nonenzymatically aminoacyl tRNA. AMP and pyrophosphate-dependent enzymatic deacylation of aminoacyl-tRNA (reverse reaction) is also stimulated by spermine. The inhibitory effect of high concentration of KC1 on aminoacylation is counteracted, by spermine. The low level of activity between pH 8.5–9.0 at 1.2 mM Mg²⁺ is restored to normal level on the addition of spermine. The inhibitory effect of high pH on aminoacylation in the presence of low concentration of Mg²⁺ is also prevntedvby spemine.     

Kinetic Origin of Substrate Specificity in Post-Transfer Editing by Leucyl-tRNA Synthetase

The intrinsic editing capacities of aminoacyl-tRNA synthetases ensure a high-fidelity translation of the amino acids that possess effective non-cognate aminoacylation surrogates. The dominant error-correction pathway comprises deacylation of misaminoacylated tRNA within the aminoacyl-tRNA synthetase editing site. To assess the origin of specificity of Escherichia coli leucyl-tRNA synthetase (LeuRS) against the cognate aminoacylation product in editing, we followed binding and catalysis independently using cognate leucyl- and non-cognate norvalyl-tRNA^Leu and their non-hydrolyzable analogues. We found that the amino acid part (leucine versus norvaline) of (mis)aminoacyl-tRNAs can contribute approximately 10-fold to ground-state discrimination at the editing site. In sharp contrast, the rate of deacylation of leucyl- and norvalyl-tRNA^Leu differed by about 10⁴-fold. We further established the critical role for the A76 3′-OH group of the tRNA^Leu in post-transfer editing, which supports the substrate-assisted deacylation mechanism. Interestingly, the abrogation of the LeuRS specificity determinant threonine 252 did not improve the affinity of the editing site for the cognate leucine as expected, but instead substantially enhanced the rate of leucyl-tRNA^Leu hydrolysis. In line with that, molecular dynamics simulations revealed that the wild-type enzyme, but not the T252A mutant, enforced leucine to adopt the side-chain conformation that promotes the steric exclusion of a putative catalytic water. Our data demonstrated that the LeuRS editing site exhibits amino acid specificity of kinetic origin, arguing against the anticipated prominent role of steric exclusion in the rejection of leucine. This feature distinguishes editing from the synthetic site, which relies on ground-state discrimination in amino acid selection.     

Direct observation of the titration of substrate carbonyl groups in the active site of alpha-chymotrypsin by resonance Raman spectroscopy

By use of resonance Raman (RR) spectroscopy, the population of the reactive carbonyl group in active acylchymotrypsins has been characterized and correlated with acyl-enzyme reactivity. RR spectra have been obtained, with a flow system and 324- and 337.5-nm excitation, at low and active pH for six acylchymotrypsins, viz., (indoleacryloyl)-, (4-amino-3-nitrocinnamoyl)-, (furylacryloyl)-, [( 5-ethylfuryl)-acryloyl]-, (thienylacryloyl)-, and [( 5-methylthienyl)acryloyl]chymotrypsin. These acyl-enzymes represent a 100-fold range of deacylation rate constants. Good RR spectral quality has enabled us to obtain the vibrational spectrum of the carbonyl group at low and active pH in each acyl-enzyme. The measured pKa of the spectroscopic changes in the carbonyl region is identical with that for the deacylation kinetics, showing that the RR carbonyl features reflect the ionization state of His-57. A carbonyl population has been observed in the active acyl-enzymes in which the carbonyl oxygen atom of the reactive acyl linkage is hydrogen-bonded in the active site. The proportion of this hydrogen-bonded population, with respect to other observed non-hydrogen-bonded species, together with the degree of polarization of the carbonyl bond, as monitored by vC = 0, has been correlated with the deacylation rate constants of the acyl-enzymes. It is proposed that the hydrogen-bonded carbonyl species is located at or near the oxyanion hole and represents the ground state from which deacylation occurs. An increase in the proportion of the hydrogen-bonded population and an increase in polarization of the carbonyl bond result in an increase in deacylation rate constant.(ABSTRACT TRUNCATED AT 250 WORDS)     

EFFECTS OF CHRONIC ETHANOL INGESTION ON BRAIN AMINOACYL-tRNA SYNTHETASES AND tRNA

The effects of chronic ethanol ingestion on the in vivo aminoacylation of brain transfer RNA (tRNA) were examined in C57BL/6J mice. A pronounced inhibition in the formation of [¹⁴C]leucy]-tRNA and [¹⁴C]phenylalanyl-tRNA was observed in the ethanol drinking mice. Properties of aminoacyl-tRNA synthetases and tRNA were examined following their separation and isolation on a DEAE-cellulose column. Synthesis of [¹⁴C]leucyl-tRNA was found to have a complete dependence on ATP and Mg²⁺. Incubations were carried out by cross-matching tRNA from control rat brain with synthetases obtained from the brains of control or ethanol-drinking mice. Under these conditions, a decreased ability for aminoacylation could be demonstrated when the source of enzyme was derived from ethanol-treated brain. The data indicate that the major effect of ethanol ingestion on the aminoacylation reaction is exerted on aminoacyl-tRNA synthetases.     

Kinetics of the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside by cellobiase of Trichoderma viride.

Cellobiase has been isolated from the crude cellulase mixture of enzymes of Trichoderma viride using column chromatographic and ion-exchange methods. The steady-state kinetics of the hydrolysis of cellobiose have been investigated as a function of cellobiose and glucose concentrations, pH of the solution, temperature, and dielectric constant, using isopropanol-buffer mixtures. The results show that (i) there is a marked activation of the reaction by initial glucose concentrations of 4 X 10(-3) M to 9 X 10(-2) M and strong inhibition of the reaction at higher initial concentrations, (ii) the log rate -pH curve has a maximum at pH 5.2 and enzyme pK values of 3.5 and 6.8, (iii) the energy of activation at pH 5.1 is 10.2 kcal mol-1 over the temperature range 5-56 degrees C, and (iv) the rate decreases from 0 to 20% (v/v) isopropanol. The hydrolysis by cellobiase (EC 3.2.1.21) of p-nitrophenyl-beta-D-glucoside was examined by pre-steady-state methods in which [enzyme]0 greater than [substrate]0, and by steady-state methods as a function of pH and temperature. The results show (i) a value for k2 of 21 S-1 at pH 7.0 (where k2 is the rate constant for the second step in the assumed two-intermediate mechanism (formula: see text), (ii) a log rate -pH curve, significantly different from that for hydrolysis of cellobiose, in which the rate increases with decreasing pH below pH 4.5, is constant in the region pH 4.5-6, and decreases above pH 6 (exhibiting an enzyme pK value of 7.3), and (iii) an activation energy of 12.5 kcal mol-1 at pH 5.7 over the temperature range 10-60 degrees C.     

The elongation factor Tu binds aminoacyl-tRNA in the presence of GDP

Escherichia coli elongation factor (EF-Tu) binds aminoacyl-tRNAs (aa-tRNA) not only in the presence of GTP but also in the presence of GDP. Complex formation leads to a protection of the aa-tRNA against nonenzymatic deacylation and digestion by pancreatic ribonuclease, as well as to a protection of EF-Tu against proteolysis by trypsin. The equilibrium constant for the binding of Phe-tRNAPheyeast for example to EF-Tu.GDP has been determined to be 0.7 X 10(5) M-1 which is 2 orders of magnitude lower than the equilibrium constant for Phe-tRNAPheyeast binding to EF-Tu.GTP. In the presence of kirromycin, aminoacyl-tRNA binding to EF-Tu.GDP is not affected as much: Phe-tRNAPheyeast is bound with an equilibrium constant of 3 X 10(5) M-1. While there is also a measurable interaction between EF-Tu.GTP and tRNA, such an interaction cannot be detected with EF-Tu.GDP and tRNA, not even at millimolar concentrations. A so far undetected complex formation between aminoacyl-tRNA and EF-Tu.GTP in the presence of pulvomycin, however, could be detected. The results are discussed in terms of the structural requirements of ternary complex formation and in the light of proofreading schemes involving A-site binding on the E. coli ribosome.     

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.     

Chemical modification as a probe of conformational changes in transfer ribonucleic acid on aminoacylation.

Treatment of Escherichia coli CA265 phenylalanyl-tRNA with 3M-NaHSO3, pH6.0, at 25 degrees C resulted in modification of four bases and in the deacylation of the charged tRNAphe. The similarity of the rates of base modification and of the deacylation of the phenylalanyl-tRNA permitted the isolation of partially modified phenylalanyl-tRNAphe and partially modified deacylated tRNAphe. The sites and extents of base modification in these fractions were determined and found to be the same as those in uncharged tRNAphe modified under identical conditions. These findings are discussed in relation to previous evidence for and against a conformational change in tRNA on its aminoacylation. The methods described should prove adaptable to study of other aminoacyl-tRNA species.     

The interaction between biologically inactive tRNA conformers and leucyl-tRNA synthetase from rabbit liver

The interaction between tRNA conformers inactive in aminoacylation and leucyl-tRNA synthetase has been investigated. Heat inactivation of the enzyme in the presence of inactive tRNA conformers is shown to lead to a marked increase of inactivation rate while active tRNA conformers, on the other hand, reveal a protecting effect. To study the properties of the enzyme complexed with different tRNA conformers limited proteolysis has been used. Active tRNA conformers are found to protect leucyl-tRNA synthetase against hydrolysis while inactive ones tend to intensify it. Inactive tRNA conformers are also shown to inhibit the aminoacylation of native tRNA in vitro. On the basis of these data biologically inactive conformers of animal tRNA are assumed to form an unproductive complex with leucyl-tRNA synthetase and the structure of the enzyme involved in such interaction is supposed to be more labile and 'extended' than that in complex with active tRNA conformers.