Citrobacter freundii tyrosine phenol-lyase: the role of asparagine 185 in modulating enzyme function through stabilization of a quinonoid intermediate

Asn185 is an invariant residue in all known sequences of TPL and of closely related tryptophanase and it may be aligned with the Asn194 in aspartate a minotransferase. According to X-ray data, in the holoenzyme and in the Mich aelis complex Asn185 does not interact with the cofactor pyridoxal 5'-phosp hate, but in the external aldimine a conformational change occurs which is accompanied by formation of a hydrogen bond between Asn185 and the oxygen a tom in position 3 of the cofactor. The substitution of Asn185 in TPL by ala nine results in a mutant N185A TPL of moderate residual activity (2%) with respect to adequate substrates, L-tyrosine and 3-fluoro-L-tyrosine, The aff inities of the mutant enzyme for various amino acid substrates and inhibito rs, studied by both steady-state and rapid kinetic techniques, were lower t han for the wild-type TPL, This effect mainly results from destabilization of the quinonoid intermediate, and it is therefore concluded that the hydro gen bond between Asn185 and the oxygen at the C-3 position of the cofactor is maintained in the quinonoid intermediate. The relative destabilization o f the quinonoid intermediate and external aldimine leads to the formation o f large amounts of gem-diamine in reactions of N185A TPL with 3-fluoro-L-ty rosine and L-phenylalanine. For the reaction with 3-fluoro-L-tyrosine it wa s first possible to determine kinetic parameters of gem-diamine formation b y the stopped-flow method. For the reactions of N185A TPL with substrates b earing good leaving groups the observed values of k(cat) could be accounted for by taking into consideration two effects: the decrease in the quinonoi d content under steady-state conditions and the increase in the quinonoid r eactivity in a beta-elimination reaction. Both effects are due to destabili zation of the quinonoid and they counterbalance each other. Multiple kineti c isotope effect studies on the reactions of N185A TPL with suitable substr ates, L-tyrosine and 3-fluoro-L-tyrosine, show that the principal mechanism of catalysis, suggested previously for the wild-type enzyme, does not chan ge, In the framework of this mechanism the observed considerable decrease i n k(cat) values for reactions of N185A TPL with L-tyrosine and 3-fluoro-L-t yrosine may be ascribed to participation of Asn185 in additional stabilizat ion of the keto quinonoid intermediate. (C) Oxford University Press.