Further Evidence for an Essential Histidyl Residue at the Active Site of Pig Liver 5-Aminolevulinic Acid Dehydratase

Abstract

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.

Production, Purification, and Characterization of a Mg2+-Responsive Porphobilinogen Synthase from Pseudomonas aeruginosa

“…One might be tempted to attribute pK a1 to a histidyl involved in catalysis. Histidine residues have been reported before to be involved in PBGS catalysis (26,27). To determine the role of the only two highly conserved histidine residues of the E. coli PBGS, both were mutated (28).…”

The influence of the vehicle on the synthesis of porphyrins after topical application of 5-aminolaevulinic acid. Implications in cutaneous photodynamic sensitization

“…ALA dehydratase (ALA‐D) activity was measured in tumour and skin homogenates at pH 6·8 in the presence of 5 mmol L −1 ALA, after 1 h of aerobic incubation 17 . Porphobilinogen (PBG) formed was quantified following the method of Mauzerall and Granick 16 .…”

“…ALA dehydratase (ALA-D) activity was measured in tumour and skin homogenates at pH 6´8 in the presence of 5 mmol L 21 ALA, after 1 h of aerobic incubation. 17 Porphobilinogen (PBG) formed was quantified following the method of Mauzerall and Granick. 16 Porphobilinogenase (PBGase) activity was measured in tumour and skin homogenates at pH 8´6 with 40 mmol L 21 PBG 18 and porphyrins were determined spectrophotometrically as described previously.…”