HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Srinivasan, R. Articles by Fisher, H. F. Search for Related Content PubMed PubMed Citation Articles by Srinivasan, R. Articles by Fisher, H. F. Social Bookmarking                      What's this?

J. Biol. Chem., Vol. 263, Issue 5, 2304-2308, 02, 1988

Mechanism of formation of bound alpha-iminoglutarate from alpha- ketoglutarate in the glutamate dehydrogenase reaction. A chemical basis for ammonia recognition

R Srinivasan, TS Viswanathan and HF Fisher
Department of Biochemistry, University of Kansas School of Medicine, Missouri.

Two mechanisms have been postulated for the formation of bound alpha- iminoglutarate intermediate during the glutamate dehydrogenase- catalyzed reductive amination of alpha-ketoglutarate; one involves the nucleophilic attack of ammonia on a covalently bound Schiff base in the enzyme-NADPH-alpha-ketoglutarate complex, and the other involves the reaction of ammonia with the carbonyl group of alpha-ketoglutarate in the ternary complex. We have measured the rates of carbonyl oxygen exchange in the complex to unambiguously distinguish between these two mechanisms. We find that the loss of label in the carbonyl oxygen- labeled ternary complex is at least 10(5) times slower than the rate of the reductive amination reaction. Therefore, the former mechanism cannot be operative. We also find that (i) the carbonyl oxygen exchange in free alpha-ketoglutarate proceeds without any significant catalysis by its gamma-carboxylate group; (ii) this exchange reaction has energy parameters which are comparable to those observed for the hydration of simple aliphatic ketones; and (iii) the carbonyl oxygen exchange in bound alpha-ketoglutarate is slower than that in the free keto acid over a wide pH range. We conclude that the oxygen exchange in the free and bound alpha-ketoglutarate must occur via a gem-diol intermediate. The observation that the enzyme inhibits the reaction of water with alpha-ketoglutarate while it catalyzes the reaction of ammonia with the same keto acid points to an extraordinary recognition of ammonia by the enzyme. We interpret this observation by assuming that the enzyme-NADPH- alpha-ketoglutarate complex exists in two forms, a predominant form which is produced rapidly upon mixing the components together and an unstable form which is produced in trace amounts from the predominant form via a gem-diol intermediate. These two forms are presumed to differ in the spatial relationship of the carbonyl group to the enzyme functional groups. The carbonyl group in the unstable form is assumed to be surrounded by the same enzyme groups as the iminium ion is in the bound iminoglutarate complex. We ascribe the remarkable catalysis of the ammonia reaction and the inhibition of the water reaction by the enzyme to the opposing interactions of the iminium and carbonyl groups with these surrounding enzyme groups.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				S. Noor and N. S. Punekar Allosteric NADP-glutamate dehydrogenase from aspergilli: purification, characterization and implications for metabolic regulation at the carbon-nitrogen interface Microbiology, May 1, 2005; 151(5): 1409 - 1419. [Abstract] [Full Text] [PDF]