The Differential Inactivation of Histidinol Dehydrogenase from Salmonella typhimurium by Sulfhydryl Reagents
Year: 1969
Type: article
Abstract: Treatment of histidinol dehydrogenase with either p-chloromercuribenzoate (CMB) or N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM), but not with N-ethylmaleimide or iodoacetic acid, produces an enzyme form which is differentially inactivated with respect to the oxidation of its two substrates, histidinol and histidinal. The binding of 2 eq of DDPM reduces activity with histidinol and histidinal as substrate to 25 and 42% of their respective initial values. Binding of 4 eq of CMB reduces activity with histidinol as the substrate to 25% of its initial value although activity measured with histidinal is not decreased. Enzymatic activity measured with histidinol as the substrate decreases as a linear function of the concentration of CMB or DDPM added, but neither reagent can totally inactivate the enzyme. During the binding of 4 eq of mercuribenzoate the rate of mercaptide bond formation appears to be proportional to the rate of inactivation measured with histidinol as the substrate. We conclude that the addition of CMB produces a uniform differential inactivation of all enzyme molecules rather than the complete inactivation of a portion of the molecules. Apparent Km values for histidinol and histidinal do not change upon differential inactivation. However, LineweaverBurk plots of velocity with respect to DPN concentration with either substrate in excess, while linear with the native enzyme, are concave downward for differentially inactivated enzyme. These effects may be due to (a) the presence of nonidentical active sites; (b) negative cooperativity between multiple active sites; or (c) an alteration in the normal reaction mechanism.
Source: Journal of Biological Chemistry
Authors: Richard E. Wolf, John C. Loper
Institution: University of Cincinnati
Language: English
Subfield: Biochemistry
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
OpenAlex ID: W1522539021