Phenylglyoxal inactivates Escherichia coli adenylate kinase by modifying a single arginine residue (Arg-88). ATP, ADP, P1,P5-di(adenosine 5')-pentaphosphate, and to a lesser extent AMP protect the enzyme against inactivation by phenylglyoxal. Site-directed mutagenesis of Arg-88 to glycine yields a modified form of adenylate kinase (RG88 mutant) closely related structurally to the wild-type protein as indicated by Fourier transform infrared spectroscopy, differential scanning calorimetry, and limited proteolysis. However, this modified protein has only 1% of the maximum catalytic activity of the wild-type enzyme and 5- and 85-fold higher apparent Km values for ATP and AMP, respectively, than the parent adenylate kinase. Arg-88, which is a highly conserved residue in all known molecular forms of adenylate kinases (corresponding to Arg-97 in muscle cytosolic enzyme), should be located inside a big cleft of the molecule, close to the phosphate-binding loop. It possibly stabilizes the transferable gamma-phosphate group from ATP to AMP in the transition state. Study holds ProTherm entries: 3114, 3115, 14157 Extra Details: Escherichia coli adenylate kinase; catalytic role; arginine;,site-directed mutagenesis; structural
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:21 p.m.
|Number of data points||9|
|Proteins||Adenylate kinase ; Adenylate kinase|
|Assays/Quantities/Protocols||Experimental Assay: ddG ; Experimental Assay: dHcal ; Experimental Assay: Tm ; Experimental Assay: dHvH ; Derived Quantity: dTm|
|Libraries||Mutations for sequence MRIILLGAPGAGKGTQAQFIMEKYGIPQISTGDMLRAAVKSGSELGKQAKDIMDAGKLVTDELVIALVKERIAQEDCRNGFLLDGFPRTIPQADAMKEAGINVDYVLEFDVPDELIVDRIVGRRVHAPSGRVYHVKFNPPKVEGKDDVTGEELTTRKDDQEETVRKRLVEYHQMTAPLIGYYSKEAEAGNTKYAKVDGTKPVAEVRADLEKILG|