TAILIEUCHUNG - Báo cáo khoa học: The mechanism of a-proton isotope exchange in amino acids catalysed by tyrosine phenol-lyase

To shed light on the mechanism of isotopic exchange of a-protons in amino acids catalyzed by pyridoxal phosphate (PLP)-dependent enzymes, we studied the kinetics of quinonoid intermediate formation for the reactions of tyrosine phenol-lyase with L-phenylalanine, L-methionine, and theira-deuterated analogues inD2O, andwe compared the results with the rates of the isotopic exchange under the same found that, in theL-phenylalanine reaction, the internal returnof thea-proton is operative, and allowing for its effect, the exchange rate is accounted for satisfactorily | Eur. J. Biochem. 271 4565-4571 2004 FEBS 2004 doi The mechanism of a-proton isotope exchange in amino acids catalysed by tyrosine phenol-lyase What is the role of quinonoid intermediates Nicolai G. Faleev1 Tatyana V. Demidkina2 Marina A. Tsvetikova1 Robert S. Phillips3 and Igor A. Yamskov1 1Nesmeyanov Institute of Organoelement Compounds Russian Academy of Sciences Moscow Russia 2Engelhardt Institute of Molecular Biology Russian Academy of Sciences Moscow Russia 3Department of Chemistry Department of Biochemistry and Molecular Biology and Center for Metalloenzyme Studies University of Georgia Athens GA USA To shed light on the mechanism of isotopic exchange of a-protons in amino acids catalyzed by pyridoxal phosphate PLP -dependent enzymes we studied the kinetics of quinonoid intermediate formation for the reactions of tyrosine phenol-lyase with L-phenylalanine L-methionine and their a-deuterated analogues in D2O and we compared the results with the rates of the isotopic exchange under the same conditions. We have found that in the L-phenylalanine reaction the internal return of the a-proton is operative and allowing for its effect the exchange rate is accounted for satisfactorily. Surprisingly for the reaction with L-methio-nine the enzymatic isotope exchange went much faster than might be predicted from the kinetic data for quinonoid intermediate formation. This result allows us to suggest the existence of an alternative possibly concerted mechanism of a-proton exchange. Keywords amino acids isotopic exchange mechanism a-proton tyrosine phenol-lyase. Pyridoxal-P-phosphate PLP -dependent lyases displaying broad substrate specificity are able to catalyze stereospecific isotope exchange of a-protons of various amino acids 1-4 including both real substrates and reversible competitive inhibitors which do not change their chemical identities under the action of the enzyme. The exchange is usually performed in heavy water and proceeds .

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