Structural and functional roles of tyrosine 78 of yeast guanylate kinase.
The hydroxyl group of Tyr-78 of yeast guanylate kinase (GK) is hydrogen-bonded to the phosphate of the bound GMP as revealed by x-ray crystallography. The structural and functional roles of Tyr-78 were evaluated by site-directed mutagenesis, kinetics, guanidine hydrochloride-induced denaturation, and nuclear magnetic resonance spectroscopy (NMR). Substitution of Tyr-78 with a phenylalanine resulted in a decrease in kcat by a factor of 131, an increase in Km(GMP) by a factor of 20 and an increase in Ki(GMP) by a factor of 18. Km(MgATP) and Ki(MgATP) were very similar to those of the wild-type (WT) GK. The conformational stability of the mutant was lower than that of the WT by 1.0 kcal/mol as measured by guanidine hydrochloride-induced denaturation. Detailed comparison of the TOCSY and NOESY spectra of the WT GK and the mutant indicated that the conformation of Y78F is little perturbed relative to that of the WT GK at the free state and the conformation of Y78F.GMP complex is also very similar to that of the WT.GMP complex. The results taken together showed that the hydrogen bond between Tyr-78 and GMP stabilizes the GK.GMP complex by 1.7 kcal/mol, the ternary complex by 1.8 kcal/mol, and the transition state by 4.6 kcal/mol. Tyr-78 is not essential for proper folding of the enzyme but it may contribute to the conformational stability. Solvent-accessible aromatic residues were identified by using the paramagnetic probe 4-hydroxy-2, 2,6,6-tetramethylpiperidine-1-oxyl. Comparison of the free and GMP-bound forms of the WT GK by NMR indicated that there are changes in conformation and dynamics upon binding of GMP.
Zhang
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Y
Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Li
Y
Y
Wu
Y
Y
Yan
H
H
D006157:Guanosine Monophosphate / Q000378:metabolism; D051528:Guanylate Kinases; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009703:Nucleoside-Phosphate Kinase / Q000737:chemistry* / Q000502:physiology; D011487:Protein Conformation; D014443:Tyrosine
D014443:Tyrosine; D006157:Guanosine Monophosphate; D009703:Nucleoside-Phosphate Kinase; D051528:Guanylate Kinases