Mechanism of adenylate kinase. Histidine-36 is not directly involved in catalysis, but protects cysteine-25 and stabilizes the tertiary structure.
Several previous reports on muscle adenylate kinase (AK) have suggested that histidine-36 (His-36) is located in the binding site of adenosine 5'-triphosphate (ATP) and is involved in catalysis. We have tested the role of His-36 using site-specific mutagenesis on chicken muscle AK expressed in Escherichia coli. Three mutant proteins (H36Q, H36N, and H36G) were obtained by substituting His-36 with glutamine, asparagine, and glycine, respectively. Steady-state kinetic studies showed that the mutants have similar kinetic properties to those of the wild-type (WT) AK, which suggested that His-36 is not directly involved in catalysis. However, His-36 is likely to interact with or protect cysteine-25 (Cys-25) on the basis of the following evidence: The crystal structure of porcine muscle AK revealed a close proximity between His-36 and Cys-25; the mutants were unstable during purification (the order of stability was WT greater than H36Q greater than H36N greater than H36G); the H36G mutant readily dimerized; the sulfhydryl groups of mutants became more reactive (WT less than H36Q less than H36N) toward 5,5'-dithiobis(2-nitrobenzoic acid). Furthermore, His-36 was found to stabilize the tertiary structure of AK on the basis of guanidine hydrochloride induced denaturation studies, which showed that the conformational stability decreases in the order WT greater than H36Q greater than H36N.(ABSTRACT TRUNCATED AT 250 WORDS)
Tian
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GC
Department of Chemistry, Ohio State University, Columbus 43210.
Sanders
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CR
Kishi
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F
Nakazawa
A
A
Tsai
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MD
D000263:Adenylate Kinase / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002645:Chickens; D003545:Cysteine; D004252:DNA Mutational Analysis; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D010770:Phosphotransferases / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D011994:Recombinant Proteins; D006639:Histidine; D010770:Phosphotransferases; D000263:Adenylate Kinase; D003545:Cysteine