The Mechanism of 6-Deoxyhexose Synthesis: V. THE RELATION OF PYRIDINE NUCLEOTIDE TO THE STRUCTURE OF THE DEOXYTHYMIDINE DIPHOSPHATE-GLUCOSE OXIDOREDUCTASE

Abstract

Abstract The deoxythymidine diphosphate d-glucose oxidoreductase from Escherichia coli is a protein of molecular weight 78,000, containing 1 mole of firmly bound DPN per mole of enzyme. Treatment of the enzyme with p-hydroxymercuribenzoate followed by removal of the mercurial with dithiothreitol releases bound DPN, and dissociates the protein into subunits of molecular weight 40,000, as determined by gel chromatography. This process is fully reversible. Inactive dimeric protein (mole wt 78,000) can also be formed by allowing the apoenzyme to react with the DPN analogue adenosine diphosphate ribose. The kinetics of reactivation of the apoenzyme with DPN suggests that the apoenzyme binds DPN with a dissociation constant of approximately 5 x 10-6 at 16° and 5 x 10-4 at 37°, followed by a slow conformational change (measured in minutes) to yield active enzyme. The apoenzyme appears to retain the substrate-binding site. Deoxythymidine diphosphate (a substrate analogue) specifically inhibits the rate of apoenzyme reactivation, and this inhibition can be reversed by the substrate deoxythymidine diphosphate glucose. The substrate analogue deoxythymidine diphosphate 6-deoxy-d-glucose reacts with enzyme to yield enzyme-DPNH and deoxythymidine diphosphate 4-keto-6-deoxy-d-glucose, the normal product of the reaction. The release of this product from enzyme-DPNH is, however, slower by at least three orders of magnitude, depending on temperature, pH, and ionic strength, than the release of the same product from enzyme-DPN in the normal catalytic reaction. The role of DPN and DPNH in the structure of the enzyme is discussed in the light of these observations.