Further evidence for an essential histidyl residue at the active site of pig liver 5-aminolevulinic acid dehydratase.
Photoxidation with methylene blue and rose bengal and chemical modification by diethylpryrocarbonate of pig liver 5-aminolevulinic acid dehydratase produced strong inactivation of the enzyme which was concentration dependent. Loss of enzyme activity by both photoxidation and ethoxyformylation was pH and time-dependent and protected by the presence of the substate and competitive inhibitors. The rate of inactivation was directly related to the state of protonation of histidyl groups, the unprotonated from being modified at a much faster rate than the protonated form. Plots of the pseudo-first order rate constants for 5-aminolevulinic acid dehydratase inactivation against pH resulted in typical titration curves showing inflection points at about pH 6.4 for methylene blue and rose bengal and 6.8 for diethylprocarbonate providing further and unequivocal evidence for the existence of critical histidyl groups at the active centre of the enzyme.
Fukuda
H
H
Department of Biochemistry, Facultad de Ciencias Exactas y Naturales, Ciudad Universitaria, Buenos Aires, Argentine.
Sopena de Kracoff
Y E
YE
Iñigo
L E
LE
Paredes
S R
SR
Ferramola de Sancovich
A M
AM
Sancovich
H A
HA
Batlle
A M
AM
D000818:Animals; D001665:Binding Sites; D004791:Enzyme Inhibitors; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008751:Methylene Blue / Q000494:pharmacology*; D010777:Photochemistry; D000623:Porphobilinogen Synthase / Q000037:antagonists & inhibitors* / Q000528:radiation effects; D012395:Rose Bengal / Q000494:pharmacology*; D013552:Swine
D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; D012395:Rose Bengal; D006639:Histidine; D000623:Porphobilinogen Synthase; D008751:Methylene Blue