Substitution of Tyr254 with Phe at the active site of flavocytochrome b2: consequences on catalysis of lactate dehydrogenation.
A role for Tyr254 in L-lactate dehydrogenation catalyzed by flavocytochrome b2 has recently been proposed on the basis of the known active-site structure and of studies that had suggested a mechanism involving the initial formation of a lactate carbanion [Lederer, F., & Mathews, F.S. (1987) in Flavins and Flavoproteins, Proceedings of the Ninth International Symposium, Atlanta, GA, 1987 (Edmondson, D.E., & McCormick, D.B., Eds.) pp 133-142, Walter de Gruyter, Berlin]. This role is now examined after replacement of Tyr254 with phenylalanine. The kcat is decreased about 40-fold, Km for lactate appears unchanged, and the mainly rate-limiting step is still alpha-hydrogen abstraction, as judged from the steady-state deuterium isotope effect. Modeling studies with lactate introduced into the active site indicate two possible substrate conformations with different hydrogen-bonding partners for the substrate hydroxyl. If the hydrogen bond is formed with Tyr254, as was initially postulated, the mechanism must involve removal by His373 of the C2 hydrogen, with carbanion formation. If, in the absence of the Tyr254 phenol group, the hydrogen bond is formed with His373 N3, the substrate is positioned in such a way that the reaction must proceed by hydride transfer. Therefore the mechanism of the Y254F enzyme was investigated so as to distinguish between the two mechanistic possibilities. 2-Hydroxy-3-butynoate behaves with the mutant as a suicide reagent, as with the wild-type enzyme. Similarly, the mutant protein also catalyzes the reduction and the dehydrohalogenation of bromopyruvate under transhydrogenation conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
INSERM U 25, CNRS UA 122, Hôpital Necker, Paris, France.
J
J
Dubois
S K
SK
Chapman
F S
FS
Mathews
G A
GA
Reid
F
F
Lederer
D001665:Binding Sites; D002384:Catalysis; D003903:Deuterium / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D006885:Hydroxybutyrates / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D042844:L-Lactate Dehydrogenase (Cytochrome); D007773:Lactates / Q000378:metabolism*; D019344:Lactic Acid; D008958:Models, Molecular; D010649:Phenylalanine; D011487:Protein Conformation; D011773:Pyruvates / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D005656:Fungal Proteins; D006885:Hydroxybutyrates; D007773:Lactates; D011773:Pyruvates; D019344:Lactic Acid; C002479:2-hydroxy-3-butynoic acid; D014443:Tyrosine; D010649:Phenylalanine; C017092:bromopyruvate; D003903:Deuterium; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome)