Ihab Rasched

Essential arginine residues in glutamate dehydrogenase.

Positively charged functional groups of amino acid residues serve as components of recognition and binding sites in the active site of enzymes which catalyze the reaction of substrates carrying negatively charged groups as carboxylate or phosphate groups. With respect to dehydrogenases arginine residues were detected to be parts of the coenzyme binding site [2-61, and lysine or arginine residues were found to be involved in the substrate binding [2,3,6-81. The participation of a positively charged group in the active site of glutamate dehydrogenase is suggested not only by the chemical nature of the substrates and the pyridine coenzymes but also by the fact that uncharged -SH reagents like iodoacetamide and N-ethylmaleimide do not alter the enzymatic activity of beef liver glutamate dehydrogenase whereas the negatively charged iodoacetic acid inactivates this enzyme irreversibly [9]. Therefore, we assume that a positively charged recognition site is essential for the orientation of the -SH reagent causing the formation of a reversible anion-protein complex before the irreversible alkylation of that cysteine residue occurs, which has been shown to be essential for the catalytic activity of glutamate dehydrogenase [9]. In addition to the functional groups necessary for the binding of the substrate and the coenzyme, positively charged functional groups are probably involved in the binding of the effecters ADP and GTP. In this paper we describe the effect of butanedione

Release of periplasmic proteins induced in E. coli by expression of an N-terminal proximal segment of the phage fd gene 3 protein

We used the enzymes β‐lactamase and alkaline phosphatase to quantitatively evaluate the release of periplasmic proteins from E. coli cells transformed by plasmids harboring gene 3 of phage fd. Different deletion mutants of gene 3 released varying fractions of the enzymes. From these results we conclude that essentially the amino‐terminal proximal part, upstream of the first glycine‐rich region but not this region itself, is responsible for the excretion of periplasmic proteins in E. coli cells expressing the gene 3 protein of phage fd.

Conformationally important histidine residue in glutamate dehydrogenase from Neurospora crassa.

The NADP-specific glutamate dehydrogenase (GDH) [l] from Neurospuru crass~ [2] like the enzyme from Candida utilis [3] (EC 1.4.1.4.) exists as an equilibrium mixture of inactive or active conformations, the ratio of the 2 forms being dependant on the pH of the environment. Native wild-type enzyme is in an active form at pH > 7.8 and in an inactive form at pH < 7.0 in the absence of substrates. The pK of this transition is near pH 7.2 for both enzymes [2,3]. The amino acid sequence of the NADP-s~ci~c CDH of N. crassa has been reported to bear regions of high homology with the same enzyme from bovine liver and chicken liver [4]. Reactive amino acids such as lysine [5,6], arginine [7] and tyrosine f8] have been identified in both mammalian and the Neuruspora enzymes with some protection against modification usually being given by the coenzyme [5-g]; it was suggested [7,8] that both the reactive arginine and tyrosine residues are in the nicotinamide-binding site of the enzyme. Here we report the presence of a group in the Neurospora crassa enzyme (NADP-specific) highly sensitive to diethylpyrocarbonate (DEP) at pH 6.0 which is usually indicative of the formation of a carboethoxyhistidine group. Evidence is presented to suggest that the histidine modified is conformationally important and that modification of this group perturbs the pH-sensitive conformational equilibrium which exists when the enzyme is in free solution at pH values close to neutrality 191.