Structural studies on the active center of α-glycerolphosphate dehydrogenase

Abstract

1. 1. In an attempt to clarify the structure of the active center of α-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), the behavior of the kinetics parameters of the enzyme as a function of pH was examined. 2. 2. The values of 6.61 found for the pK 1 of the free enzyme and 6.72 for the pK 1 of the enzyme-substrate complex suggested the presence of atleast one histidyl residue at the active center. 3. 3. Photo-oxidation of the enzyme in the presence of Rose Bengal as sensitizer inactivated the enzyme. This inactivation was dependent on the destruction of a group or groups having a pK a of 7.3. 4. 4. Chemical modification of the enzyme with diazo- i-H-tetrazole, under conditions where neither bisazohistidine nor monoazo- or bisazotyrosine could be detected, showed that the conversion of 1.07 histidine to the monoazo product led to a 50% inactivation. When 1.77 monoazohistidine residues had been formed, the level of inactivation was 94%. Complete inactivation was attained when 2.14 histidines had been converted to the monoazoderivative. 5. 5. The results reported here strongly suggest that two histidine residues at the active center of the α-glycerolphosphate dehydrogenase are critical for the activity of this enzyme.