STRUCTURE-FUNCTION RELATIONSHIPS OF GLUTAMATE SYNTHASE
Abstract
The structure, function and mechanism of Escherichia coli glutamate synthase were investigated. A radioimmunoassay established the NH(,3)-dependent activity was not due to contaminating glutamate dehydrogenase. Negative cooperativity was observed in 2-oxoglutarate saturation of the NH(,3)- but not the glutamine-dependent activity. pH independent "half sites" binding was observed for 2-oxoglutarate in the absence of glutamine (K(,d) < 0.5 (mu)M). Binding of 2-oxoglutarate in the presence of glutamine was pH-dependent. Binding varied from 0.43 eq. per (alpha)(beta) protomer (K(,d) 0.5 (mu)M) at pH 6.7 to 2.3 eq. (K(,d) 39.4 (mu)M) at pH 9.0 with binding of 0.97 eq.per protomer (K(,d) 2.7 (mu)M) at pH 7.5. It is suggested that binding of glutamine promotes a conformational change that opens a distinct 2-oxoglutarate site which is utilized in the glutamine-dependent reaction. Binding of NADP('+) was to a single dinucleotide site per (alpha)(beta) protomer (K(,d) 5 (mu)M). Arginyl residue modification by phenylglyoxal and butanedione inactivated diaphorase, glutamine- and NH(,3)-dependent activities of glutamate synthase. NADP('+) protected against arginyl modification and modification abolished NADP('+) binding. Glutamate synthase has a single dinucleotide binding site per protomer which is utilized in the diaphorase, glutamine- and NH(,3)-dependent activities. Chemical modification with pyridoxal 5'-phosphate inactivated glutamine-dependent but not NH(,3)-dependent activities of glutamate synthase and anthranilate synthase. Inactivations were ascribed to Schiff base formation at essential lysyl residues in the glutamine active sites. The Schiff bases were labile but could be stabilized by HCN addition or reduction. Pyridoxal 5'-phosphate modification prevented binding of glutamine to glutamate synthase but was shown to reduce the reactivity of an essential cysteinyl residue in anthranilate synthase.
Degree
Ph.D.
Subject Area
Biochemistry
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