Abstract
The glutamate dehydrogenase-NADPH-alpha-ketoglutarate complex, an active intermediate on the reaction pathway has a number of unusual properties: 1) it is the only blue-shifted natural complex of this enzyme; 2) it has an anomalously slow rate of dissociation; 3) its off-rate shows a substantial pH-independent D2O solvent isotope effect not exhibited by any other ternary complex of this enzyme; and 4) it has an unusually large enthalpy of interaction parameter. These properties must be ascribable to at least one of the two possibilities conferred on the complex by the presence of the alpha-carbonyl group of alpha-ketoglutarate; the ability to engage in carbonyl addition reactions; and/or the ability to form a specific hydrogen bond. Oxalylglycine, a competitive inhibitor of alpha-ketoglutarate in this enzyme-catalyzed reaction, provides a means of discriminating between these two modes of action. The structure of oxalylglycine provides a dicarboxylic compound which has the same intercarboxylate proton distance and has a carbonyl group in a position spatially analogous to that of alpha-ketoglutarate. Its carbonyl group, however, is that of an amide group and cannot, therefore, engage in carbonyl addition reactions, but can hydrogen bond. Therefore, any effects observed with both oxalylglycine and alpha-ketoglutarate must be ascribed to formation of specific alpha-carbonyl hydrogen bonding, whereas any effects observed with alpha-ketoglutarate alone must be due to an alpha-carbonyl addition reaction. We have used this logic to test the source of the four phenomena listed above. In each case, oxalylglycine and alpha-ketoglutarate showed the same effect. Therefore, we conclude that all four phenomena are in fact due to the formation of a specific alpha-carbonyl hydrogen bond and that the specific carbonyl addition reaction between alpha-ketoglutarate and an enzyme lysine group, postulated in one proposed catalytic mechanism, does not occur.
Highlights
Theglutamate dehydrogenase-NADPH-a-ketoglu- complex in this reaction for two reasons: 1) it occupies the tarate complex, an active intermediateon the reaction position of minimum free energy on the reaction pathway, 2)
Oxalylglycine can serve as a discriminantbetween the two possible functions of the carbonyl group of a-ketoglutarate:any phenomenon caused by both a-ketoglutarate and oxalylglycinemust be due to theironly common property-hydrogen bonding, any phenomenon caused by a-ketoglutarate alone must be ascribed to its unique capability-a carbonyl addition reaction
It can be seen that the E-NADPH-oxalylglycine complex does have the same blue-shifted 340 nm band as that which characterizes the spectrum of the E-NADPH-a-ketoglutarate complex, in contrast to the red-shifted spectra seen for all other complexes of this enzyme
Summary
Vol 262, No 24, Ieaue of August 25,pp. 116841-91817687 Printed in 2) it has an anomalously slow rate of dissociation; 3) its off-rate shows a substantial pH-independent DaO solvent isotope effect not exhibited by any other ternary complex of this enzyme; and 4) it hasan unusually large enthalpy of interaction parameter These properties must be ascribable to at least one of the two possibilities conferred on the complex bythe presence of the a-carbonyl groupof a-ketoglutarate; the ability (1h E-NADPH-a-ketoglutarate exhibits a number of unusual properties which are not sharedby any otherknown complex of glutamate dehydrogenase: 1)The 340 nm peak of NADPH is blue-shifted to 332 nm in the E-NADPH-a-ketoglutarate complex. Tures of the otherligands alters the conformation of the fivecarbon dicarboxylate skeleton considerably It is to be expected, that the a-carbonyl oxygen atom might be able to form hydrogen bonds with enzyme groups which are. In both molecules the a-carbon atom is sp2hybridized, they have the same intercarboxylate-proton distance, through a Corning 0 - 5 2 band-pass filter having a cutoff wavelength of 340 nm and a Schott UG-5 filter having a cutoff wavelength 2 600 nm
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