Motohiro Iwatsubo

Evidence for two nicotinamide binding sites on L-glutamate dehydrogenase

Abstract Circular dichroism saturation in the nicotinamide band of NADH, provides direct evidence for the binding of two nicotinamide rings per protomer of L-glutamate dehydrogenase. These two binding sites are titrated by NADH in the presence of both the substrate (L-glutamate) and an allosteric effector (GTP or Zn2+) while only one reacts in the absence of the effector. We suggest that the second binding site, not accessible to NADPH, is demasked by a conformational change of the protein induced by the allosteric effector.

Antico-operative binding of bacterial and mammalian initiator tRNAMet to methionyl-tRNA synthetase from escherichia coli.

The reaction scheme of methionyl-tRNA synthetase from Escherichia coli with the initiator tRNAsMet from E. coli and rabbit liver, respectively, has been resolved. The statistical rate constants for the formation, kR, and for the dissociation, kD, of the 1:1 complex of these tRNAs with the dimeric enzyme have been calculated. Identical kR values of 250 μm−1 s−1 reflect similar behaviour for antico-operative binding of both tRNAsMet to native methionyl-tRNA synthetase. Advantage was taken of the difference in extent of tryptophan fluorescence-quenching induced by the bacterial and mammalian initiator tRNAsMet to measure the mode of exchange of these tRNAs antico-operatively bound to the enzyme. Analysis of the results reveals that antico-operativity does not arise from structural asymmetric assembly of the enzyme subunits. Indeed, both subunits can potentially bind a tRNA molecule. Exchange between tRNA molecules can occur via a transient complex in which both sites are occupied. Either strong and weak sites reciprocate between subunits on the transient complex or occupation of the weak site induces symmetry of this complex. While in the present case, these two alternatives are kinetically indistinguishable, they do account for the observation that, upon increasing the concentration of the competing mammalian tRNA, the rate of exchange of the E. coli initiator tRNAMet is enhanced, due to its faster rate of dissociation from the transient complex. Finally, it has been verified that in the case of the trypsin-modified methionyl-tRNA synthetase which cannot provide more than one binding site for tRNA, exchange of enzymebound bacterial tRNA by mammalian tRNA does proceed to a limiting rate independent of the mammalian tRNA concentration present in the solution.

Biosynthetic incorporation of cobalt into yeast alcohol dehydrogenase

Many enzymes are metalloproteins. Numerous biochemical and biophysical studies have been done to collect information on the role of the metal in the enzymatically catalysed reaction. When it is possible to remove the metal reversibly and to obtain an inactive apoenzyme, the effect of the in vitro recombination of the apoenzyme, with one or several metals, can be studied; among other investigations, this has been done for bovine pancreatic carboxypeptidase [l-3] , carbonic anhydrase [4-61, yeast D-LDH [7-l l] ; from a more biological point of view, a related problem is the study of in vivo effects of different metals on the synthesis of some metalloenzymes and on their properties; various questions arise, such as: is it possible to incorporate in vivo in a metalloenzyme a metal other than the one present in the molecule under natural conditions of synthesis? If so, it becomes of interest to study the modifications introduced by the substitution in vivo of the metallic cofactor of the enzyme during biosynthesis in cells. This investigation can be carried out in the case of microorganisms for which the chemical composition (metal and salts) of growth media can be controlled, and when growth occurs in spite of deprivation or enrichment in some metals. Such a study has been performed for yeast D-LDH which is a zincflavoenzyme [7-l l] ; its inactive apoenzyme can be prepared [7] , and has chelating properties [ lo] . When yeast was grown in synthetic media deprived or enriched with Zn, Co, Mn, it has been shown that, in the absence of zinc and in the presence of cobalt,

Beef liver L-Glutamate dehydrogenase mechanism: presteady state study of the catalytic reduction of 2.oxoglutarate by NADPH.

The mechanism of the reduction of 2.oxoglutarate by NADPH and addition of NH4+ catalyzed by L-glutamate dehydrogenase was investigated with the stopped flow technique, following dihydronicotinamide absorbance and fluorescence changes. We make conspicuous, as in the reverse reaction, the existence of prestationnary events, the characteristics of which strongly depend on the ligand addition order. If the E-NADPH-2.oxoglutarate complex is preformed, saturated with regard to each ligand, there is a burst associated with the oxidation of 0.7 mole of NADPH per mole of protomer, a first order process with a velocity constant of 300 ± 50 sec−1. If this complex is not preformed, the burst has reduced amplitude and velocity constants, differences that suggest an isomerization step following the binding processes. Whichever the incubation order, the later evolution follows an exponential decrease towards equilibrium with a velocity constant depending on the ligand concentration up to an extrapolated value of 27 sec−1, a step corresponding to the dissociation of the reaction products.