THE PH-DEPENDENCE AND MODIFICATION BY DIETHYL PYROCARBONATE OF ISOCITRATE LYASE FROM PHYCOMYCES-BLAKESLEEANUS

We determined the variation with pH of the kinetic parameters for the isocitrate cleavage reaction catalyzed by Phycomyces isocitrate lyase, with the aim of elucidating the role played by ionising amino acid re sidues in binding and catalysis. The log V-maxpH profile shows that th e enzyme possesses two ionising groups with pK values of 6.1 and 8.3. The first group is also observed in the V-maxpH/K-mpH and pK(mpH) prof iles, so this group is involved in catalysis. The last two profiles ex hibit a similar pK value of 16 on the basic side, which represents the sum of the pK values for two ionising groups with pK values that diff er by less than two pH units. Diethyl pyrocarbonate inactivated isocit rate lyase from Phycomyces with a second-order rate constant of 18.58 M(-1) s(-1) (at pH 6.0 and 20 degrees C). The difference spectra of th e modified enzyme revealed an absorption maximum at 242 nm, characteri stic of N-carbethoxyhistidine isocitrate lyase. No trough at around 28 0 nm due to O-carbethoxytyrosine is observed. Quantification of the in crease in absorbance to 242 nm due to N-carbethoxyhistidine showed tha t ten histidine residues/active site were modified during total inacti vation. However, only one of them was essential for catalysis. Treatme nt of the partially inactivated enzyme with hydroxylamine led to recov ery of a substantial part of the original activity. The reactivity of isocitrate lyase towards diethyl pyrocarbonate declined with pH, follo wing a titration curve for a group of pK 6.1. The presence of substrat e decreased the rate of inactivation. Data-protection analyses indicat e that the reactive histidine residues are within the active site of t he enzyme.