Effect of dyethylpyrocarbonate on the conformation and enzymic activity of D-glyceraldehyde-3-phosphate

Ovádi, Judit and Keleti, Tamás (1969) Effect of dyethylpyrocarbonate on the conformation and enzymic activity of D-glyceraldehyde-3-phosphate. ACTA BIOCHIMICA ET BIOPHYSICA ACADEMIAE SCIENTIARUM HUNGARICAE, 4 (4). pp. 365-378. ISSN 0001-5253

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Abstract

In denatured D-glyceraldehyde-3-phosphate dehydrogenase all (11 per subunit) histidyl residues react with diethylpyrocarbonate. Six residues react if 100 mole equi valents and four if 25 mole equivalents per subunit of this reagent are used. The apparently first order rate constant of decrease in enzymic activity is prac tically identical with the rate constant of the formation of carbethoxy-histidyl groups in the presence of 25 moles diethylpyrocarbonate per subunit. D-glyceraldehyde-3-phosphate dehydrogenase, which is completely inactivated by the formation of 4 carbethoxy-histidyl bonds per subunit in the absence of substrates, may be partially protected from inactivation if treatment with diethylpyrocarbonate is carried out in the presence of NAD and/or phosphate. Moreover, incubation with substrates and mercaptoethanol partially reactivates the fully inactive carbethoxy- enzyme. However, in each case the same number of carbethoxy-histidyl groups are formed. The peptide containing two out of four carbethoxylated histidyl residues was isolated and analyzed. The dissociation of two out of the four firmly bound NAD molecules increases after carbéthoxylation of the enzyme. The fluorescence of the carbethoxylated enzyme increases indicating the loosening of the steric structure of D-glyceraldehyde-3-phos- phate dehydrogenase. These results suggest that carbéthoxylation of four histidyl residues per subunit alters the conformation of the enzyme. Some of the modified residues may be in the neighbourhood of the active centre, however, probably are not directly involved in the catalytic activity.

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