Mechanism of adenylate kinase. Structural and functional demonstration of arginine-138 as a key catalytic residue that cannot be replaced by lysine.
Replacement of the arginine-138 of adenylate kinase (AK) by lysine or methionine resulted in a decrease in kcat by a factor of 10(4), increases in Km by a factor of 10-20, and relatively little changes in dissociation constants. Proton nuclear magnetic resonance (NMR) studies were then undertaken to obtain structural information for quantitative interpretation of the kinetic data. Since the lysine mutant (R138K) represents a conservative mutation with surprisingly large effects on kinetics, structural studies were focused on the wild type (WT) and R138K. The results and conclusions are summarized as follows: (i) The aromatic spin systems of WT and R138K were assigned from total correlated spectroscopy (TOCSY). Comparison of the chemical shifts of aromatic protons, one-dimensional spectra, TOCSY, and nuclear Overhauser enhanced spectroscopy (NOESY) indicated that the conformation of R138K was almost unperturbed relative to that of WT. Thus Arg-138 is not important for the tertiary structure. (ii) Proton NMR titrations with AMP and MgATP suggested that substrate binding affinities and substrate-induced conformational changes are nearly identical between WT and R138K. Thus arginine-138 should not be involved in stabilizing the first substrate in the binary complex. (iii) Notable differences were observed between the proton NMR spectra of the WT and R138K complexes with the reaction mixture, which agrees with the perturbation in the Km values of R138K. The differences were analyzed in detail by using a "static reaction mixture'--p1, p5-bis(5'-adenosyl)pentaphosphate (MgAP5A). The aromatic spin systems of WT + MgAP5A and R138K + MgAP5A were partially assigned from various two-dimensional spectra.(ABSTRACT TRUNCATED AT 250 WORDS)
Yan
H G
HG
Department of Chemistry, Ohio State University, Columbus 43210.
Shi
Z T
ZT
Tsai
M D
MD
D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000263:Adenylate Kinase / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D006801:Humans; D007700:Kinetics; D008239:Lysine; D008274:Magnesium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008715:Methionine; D008969:Molecular Sequence Data; D013329:Structure-Activity Relationship
D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D001120:Arginine; D008715:Methionine; D000263:Adenylate Kinase; D008274:Magnesium; D008239:Lysine