Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides: ligand-induced conformational changes.
Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is inactivated by trypsin, chymotrypsin, pronase E, thermolysin, 4.0 M urea, and by heating to 49 degrees C. It is protected, to varying degrees, against all these forms of inactivation by glucose 6-phosphate, NAD+, and NADP+. When these ligands are present at 10 times their respective KD concentrations, protection by NAD+ or glucose 6-phosphate is substantially greater than protection by NADP+. A detailed analysis was undertaken of the protective effects of these ligands, at varying concentrations, on proteolysis of glucose-6-phosphate dehydrogenase by thermolysin. This study confirmed the above conclusion and permitted calculation of KD values for NAD+, NADP+, and glucose 6-phosphate that agree with such values determined by independent means. For NADP+, two KD values, 6.1 microM and 8.0 mM, can be derived, associated with protection against thermolysin by low and high NADP+ concentrations, respectively. The former value is in agreement with other determinations of KD and the latter value appears to represent binding of NADP+ to a second site which causes inhibition of catalysis. A Ki value of 10.5 mM for NADP+ was derived from inhibition studies. The principal conclusion from these studies is that NAD+ binding to L. mesenteroides glucose-6-phosphate dehydrogenase results in a larger global conformational change of the enzyme than does NADP+ binding. Presumably, a substantially larger proportion of the free energy of binding of NAD+, compared to NADP+, is used to alter the enzyme's conformation, as reflected in a much higher KD value. This may play an important role in enabling this dual nucleotide-specific dehydrogenase to accommodate either NAD+ or NADP+ at the same binding site.
Kurlandsky
S B
SB
Department of Biology, Syracuse University, New York 13244.
Hilburger
A C
AC
Levy
H R
HR
D004789:Enzyme Activation / Q000187:drug effects; D019298:Glucose-6-Phosphate; D005954:Glucosephosphate Dehydrogenase; D005958:Glucosephosphates; D006358:Hot Temperature; D007934:Leuconostoc / Q000187:drug effects / Q000201:enzymology*; D008024:Ligands; D009243:NAD; D009249:NADP; D010447:Peptide Hydrolases; D011487:Protein Conformation / Q000187:drug effects*; D014508:Urea
D005958:Glucosephosphates; D008024:Ligands; D009243:NAD; D009249:NADP; D019298:Glucose-6-Phosphate; D014508:Urea; D005954:Glucosephosphate Dehydrogenase; D010447:Peptide Hydrolases