Dissecting the reaction mechanism of sheep liver 6-phosphogluconate dehydrogenase.

In the second part of the research, three additional mutants, S 128A, H 186A and N187A were characterized in the same manner as the K183 mutant enzymes. The decrease in the activity compared to the wild type enzyme is about 200-fold for the H 186A and N 187A mutant enzymes, but only 12-fold for the S 128A mutant enzyme. Dissociation constant for 6PG from the E:NADP:6PG complex (K6PG) is increased by around 6-fold for both S 128A and H 186A and 16-fold for N 187A. Product inhibition studies by NADPH give a dissociation constant for the E:NADPH complex (Kis) that is increased by 5- to 6-fold for the S 128A and H 186A mutant enzymes at nonsaturating 6PG. No significant change is found in Kis value for the N 187A mutant enzyme. The primary deuterium isotope effects decrease for S 128A and H 186A, and increase in the case of N 187A compared to those of the wild type enzyme. The kinetic data suggest that all of the three enzyme side chains are responsible for binding the substrates, and that both S 128 and H 186 play an important role in the decarboxylation process, while N187 facilitates the hydride transfer step.

Author dc:creator

• Zhang, Lei.

Advisor dc:contributor.advisor

• Cook, Paul F.,

• Enzymes Synthesis.
• Pentose phosphate pathway.
• Proteins Research.
• Dehydrogenases.
• Chemistry, Biochemistry.

Handle dc:identifier.uri

http://hdl.handle.net/11244/6019

OAI record oai:identifier

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