Structure-Function Relationship of Glucose 6-Phosphate Dehydrogenase from Leuconostoc Mesenteroides

Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)




H. Richard Levy


Lysine, ligand binding, conformational transitions, fluorescence reporter group, extrinsic probe

Subject Categories



The previous procedure for the isolation of glucose 6-phosphate dehydrogenase (G6PD) from Leuconostoc mesenteroides was modified to allow the preparation of large quantities of homogeneous enzyme suitable for structural studies. The enzyme was shown to consist of two identical subunits containing N-terminal valine and C-terminal glycine.

Three aspects were examined that probe the relationship between structure and function of L. mesenteroides G6PD. First, the enzyme was covalently labelled with a phosphopyridoxyl group at a unique lysine residue. Previous work had suggested that this lysine is located at the active site and that it participates in binding G6P {Milhausen and Levy, Eur., J. Biochem. 50 (1975), 453-461}. Two pyridoxyl peptides, DIIA and DIIB, were isolated from the tryptic digest of the modified enzyme. The amino acid sequence of each peptide revealed that the sequence of the pyridoxyl binding site was Phe-Leu-Leu-Lys(Pxy)-Ser-Pro-Ser-Tyr-(Asp, Val)-Lys. This is the first report of sequence information from the active site of any G6PD.

Support for the role of the modified lysine is provided in the second part of this dissertation, in which three fluorescent probes were exploited to provide information on ligand binding and conformational transitions that the enzyme undergoes.

First, the intrinsic protein fluorescence was quenched by binding various ligands to the enzyme. These measurements permitted the monitoring of overall conformational changes of the enzyme upon ligand binding and calculation of binding constants.

Second, the covalently bound phosphopyridoxyl group in the modified enzyme served as a fluorescence reporter group to monitor the conformational changes produced by NAD('+) or NADP('+) and to calculate their binding constants. The results of these experiments showed that pyridoxylation promotes a conformational change similar to that produced by NAD('+) or G6P (see below). Binding of NAD('+) to pyridoxyl enzyme is accompanied by a smaller conformational change than that seen when it binds to the native enzyme. Conversely, NADP('+) binding leads to a larger conformational change in the pyridoxylated than in the native enzyme. This was supported by the fact that the dissociation constant for NAD('+) is lower, and for NADP('+) is higher, for pyridoxyl enzyme than for native enzyme.

Third, the fluorescence of S-NADPH was used as an extrinsic probe of the coenzyme binding region of the enzyme. The fluorescence properties of S-NADPH are reported for the first time. ...