Date of Award

Summer 1976

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry & Biochemistry

Program/Concentration

Chemical Sciences

Committee Director

James H. Yuan

Committee Member

Thomas O. Sitz

Committee Member

R. O. Carter

Call Number for Print

Special Collections LD4331.C45R67

Abstract

The properties of the coenzyme, NAD, binding site of chicken muscle L-∝-glycerophosphate dehydrogenase were studied. Adenosine monophosphate, adenosine diphosphate and adenosine diphosphoribose were sha.vn to inhibit the enzyme competitively with respect to NAD. The presence of adenosine, pyrophosphate and ribose regions in the coenzyme binding site of the enzyme was suggested by the inhibitor constants obtained for these compounds.

Disodium monoalkyl phosphates, n-butyl to n-dodecyl phosphate, inclusive, were also shown to inhibit the L-∝-glycerophosphate dehydrogenase catalized reaction competitively with respect to NAD. A positive chain length effect was observed in the binding of these compounds to the enzyme, suggesting the presence of a hydrophobic region in the coenzyme binding site of the enzyme. Multiple inhibition studies demonstrated the simultaneous binding of the inhibitor pair adenosine monophosphate and disodium n-heptyl phosphate. However, mutual exclusion was observed in the multiple inhibition studies with inhibitor pairs of disodium n-heptyl phosphate and disodium n-undecyl phosphate, and adenosine diphosphoribose and disodium n-heptyl phosphate. These results suggested the presence of a hydrophobic region in or nearby the ribose binding region of the coenzyme binding site of the enzyme.

Fluorescence quenching was used to study the properties of the binding of coenzyme-competitive inhibitors to L-∝-glycerophosphate dehydrogenase. The binding of adenosine diphosphoribose and 3-aminopyridine adenine dinucleotide to chicken muscle ∝-glycerophosphate dehydrogenase in the absence of substrate was observed to be anticooperative and in the presence of substrate noncooperative. However, the binding of either adenosine monophosphate or disodium n-dodecyl phosphate was found to be noncooperative whether in the presence or absence of substrate. Results indicate that the binding of coenzyme-competitive inhibitors that occupy an area in the coenzyme binding site smaller than adenosine diphosphoribose cannot provide the steric requirements necessary for the normal type of cooperativity seen with the analog of NAD, 3-aminopyridine adenine dinucleotide. The change from anticooperativity in the absence of substrate to noncooperativity in the presence of substrate for 3-aminopyridine adenine dinucleotide and adenosine diphosphoribose results from decreased interactions between the NAO binding region on each subunit through formation of the ternary complex.

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DOI

10.25777/g4qt-c462

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