PYRIDOXAL-5'-PHOSPHATE-SENSITIZED PHOTOINACTIVATION OF TRYPTOPHANASE AND EVIDENCE FOR ESSENTIAL HISTIDYL RESIDUES IN THE ACTIVE-SITES

Tryptophanase of Escherichia coli B/It7‐A was rapidly inactivated by visible light irradiation in the presence of pyridoxal 5′‐phosphate (pyridoxal‐P). This photoinactivation followed pseudo‐firstorder kinetics and did not cause a change in molecular size of enzyme. The essential importance of the active‐site‐bound pyridoxal‐P was demonstrated by the following observations: (a) the same photoinactivation rate between in the presence and absence of large excess unbound pyridoxal‐P; (b) a structural requirement of photosensitizer for 4‐aldehyde and 5′‐phosphate groups; (c) a correlation of the effect of monovalent cations on the photoinactivation rate with their cofactor activity, that is, rapid inactivation with K+, NH+4 and Rb+ (active as cofactor in enzyme catalysis) and much slower rate with Li+, Na+ and Cs+ (inactive cations) as well as without inorganic monovalent cations was seen. Both l‐tryptophan (substrate) and l‐alanine (competitive inhibitor) markedly decreased the rate of photoinactivation. A half‐maximal rate of inactivation was obtained at pH 7.2 on pyridoxal‐P‐sensitized photoinactivation of holoenzyme and at pH 7.3 on methylene‐blusensitized photoinactivation of apoenzyme, which are close to the pKa value of the histidyl residue. Ammo acid analysis showed that photoinactivation was accompanied by disappearance of only one histidyl residue per subunit. Although lysyl residues also decreased in the presence of a large excess of pyridoxal‐P (200 μM), no appreciable decrease of lysyl residues was observed at a lower concentration of pyridoxal‐P (5 μM). No other amino acid residues were modified by irradiation in either case. Thus, it can be concluded that tryptophanase has one essential histidyl residue per active site and this residue comes in close proximity to the coenzyme in the catalytically active holoenzyme. Copyright © 1979, Wiley Blackwell. All rights reserved