Chemical modification of bacteriophage T4 deoxynucleotide kinase. Evidence of a single catalytic region.
The mechanism underlying the unusual specificity of bacteriophage T4 deoxynucleotide kinase, which catalyzes the phosphorylation of 5-hydroxymethyldeoxycytidylate, dTMP, and dGMP, has been investigated by chemical modification of the protein. Pyridoxal 5'-phosphate inactivates deoxynucleotide kinase by modifying a single lysine out of the 17 per monomer. Lysine 10 has been tentatively identified as the site of modification, although the possibility of mutually exclusive reactive residues has not been eliminated. Diethylpyrocarbonate also inactivates the enzyme, suggesting that histidine plays a role in catalytic function. With either reagent, the three activities are lost at equal rates, supporting the contention that one active site is responsible for the exclusive phosphorylation of three dissimilar deoxynucleotides. These studies also identify two distant regions of the primary sequence that are likely to be closely associated in the active region of the folded protein.
McCollum-Pratt Institute, Johns Hopkins University, Baltimore, Maryland 21218.
G S
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Brush
M J
MJ
Bessman
D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000201:enzymology*; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003843:Deoxycytidine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D009703:Nucleoside-Phosphate Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D013379:Substrate Specificity; D013938:Thymidine Monophosphate / Q000378:metabolism
D003848:Deoxyguanine Nucleotides; D003843:Deoxycytidine Monophosphate; C079259:5-hydroxymethyldeoxycytidylic acid; D013938:Thymidine Monophosphate; D006639:Histidine; D011732:Pyridoxal Phosphate; C007257:2'-deoxyguanosine 5'-phosphate; D009703:Nucleoside-Phosphate Kinase; D008239:Lysine; D004047:Diethyl Pyrocarbonate