The Effects of Immobilization on Structure and Substrate Activation of Glutamate Dehydrogenase

Citation

O'Connor, Kim Claire (1987) The Effects of Immobilization on Structure and Substrate Activation of Glutamate Dehydrogenase. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/qavk-kj50. https://resolver.caltech.edu/CaltechETD:etd-03042008-094030

Abstract

Structure and substrate activation of bovine liver glutamate dehydrogenase were investigated when the enzyme was in its native state or was covalently bound via primary amino groups to a solid Sepharose bead. In this research, the enzyme was covalently bound directly to CNBr-activated Sepharose 4B or indirectly, via a six-carbon spacer arm, to CH-activated Sepharose 4B. Electron spin resonance spectroscopy was employed to study structure. This study required spin labeling glutamate dehydrogenase with either 2,2,6,6-tetramethyl-4-oxopiperidine-1-oxyl or 4((4-(chloromercurio)benzoyl)amino)-2,2,6,6-tetramethyl-1-piperidinyloxy. Substrate activation of the deamination of glutamate by NAD was visible in Lineweaver-Burk plots of inverse rate vs. inverse substrate concentration.

The electron spin resonance spectrum of glutamate dehydrogenase spin labeled with 2,2,6,6-tetramethyl-4-oxopiperidine-1-oxyl was a composite consisting primarily of two populations of spin labels with outer peak separations of 33 gauss (population A) and 69 gauss (population B). For the native enzyme in solution, population A accounted for 10% of the spectrum. This percentage increased to 21 when the active site was saturated with substrates during spin labeling and increased to 34 when the enzyme was immobilized to either CNBror CH-activated Sepharose. The roles of aspecific labeling and structural heterogeneity in these spectral changes were discussed. Modification of the native and immobilized enzyme with pyridoxal 5'-phosphate characterized the binding site(s) for the spin label. Deconvolution of experimental spectra into populations A and B divulged a subpopulation of labels with a mobility unlike those of the two principal components. Linear combinations of the deconvoluted line shapes for populations A and B successfully represented all spectra of glutamate dehydrogenase from this and previous investigations.

Conformational change in glutamate dehydrogenase was investigated by spin labeling the enzyme at cysteine 319 with 4((4-(chloromercurio)benzoyl)-amino)-2,2,6,6-tetramethyl-1-piperidinyloxy. Electron spin resonance spectra of spin-labeled glutamate dehydrogenase showed that immobilization of the enzyme on CNBr-activated Sepharose 4B suppressed conformational change induced by GTP and NADPH and profoundly altered that induced by aketoglutarate. Moreover, the spectrum of the immobilized enzyme provides evidence for structural heterogeneity at cysteine 319.

Covalently binding glutamate dehydrogenase to CNBr- and CH-activated Sepharose 4B altered the substrate activation pattern of the biocatalyst. Specifically, the sharp discontinuity, which characterizes Lineweaver-Burk plots of free enzyme kinetics with NAD as the varied substrate, became elongated when the biocatalyst was attached to either support. The elongated transition region contains two inflection points and resembles substrate activation of several other allosteric oligomers. Under the same experimental conditions, glutamate induced varying degrees of abrupt activation in immobilized glutamate dehydrogenase and inhibited the enzyme in solution. The intensity of this activation is inversely proportional to the rate constant of the biocatalyst-Sepharose conugate. These modified activation patterns may be due in part to increased structural heterogeneity and altered conformational change. The above kinetic results are free of mass transport effects: the external effectiveness factor was unity, the observable Thiele modulus was less than 2.0 • 10^-4, and reaction- generated pH change was less than 0.01 for all data reported in this paper.

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