The roles of Tyr70 and Tyr225 in aspartate aminotransferase assessed by analysing the effects of mutations on the multiple reactions of the substrate analogue serine o-sulphate.
Aspartate aminotransferase catalyses multiple reactions of the glutamate analogue, serine O-sulphate. The predominant reaction is beta-elimination of sulphate to give aminoacrylate (kcat = 13 s-1 for the Escherichia coli enzyme) which may either hydrolyse to pyruvate and ammonia, or react covalently with the enzyme and inactivate it (kinact = 1.1 x 10(-3) s-1). Serine O-sulphate also undergoes a transamination reaction that converts the enzyme to its pyridoxamine form (kcat = 0.11 s-1). Tyr70 and Tyr225, each of which forms a hydrogen bond with the coenzyme, were substituted with methionine and phenylalanine, respectively. The Y225F mutation does not affect beta-elimination but reduces the rates of transamination and inactivation about 70-fold and 3-fold, respectively. Apparently, Tyr225 is not essential for the steps leading to and including abstraction of the proton from C alpha of the substrate. It is argued that the Y225F mutation interferes with ketimine hydrolysis. The Y70M mutation affects all three reactions, beta-elimination being about fourfold slower, transamination 340-fold slower, and inactivation being 1.4 times faster than in the wild-type enzyme. It is proposed that a hydrogen bond from Tyr70 positions Lys258 for protonation of the quinonoid intermediate at C4' and that, although the full kinetic contribution of this interaction is only revealed in the multiple reactions of serine O-sulphate, the same interaction is equally important in increasing the reaction specificity for transamination of the natural substrates.
Birolo
L
L
School of Molecular and Medical Biosciences, University of Wales, Cardiff, UK.
Sandmeier
E
E
Christen
P
P
John
R A
RA
D000586:Amination; D001219:Aspartate Aminotransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008715:Methionine; D009154:Mutation; D010649:Phenylalanine; D012694:Serine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000378:metabolism*
D014443:Tyrosine; D012694:Serine; D010649:Phenylalanine; C036040:serine O-sulfate; D008715:Methionine; D001219:Aspartate Aminotransferases