Abstract
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.
Highlights
The mechanismunderlying the unusual specificity of bacteriophage T4 deoxynucleotide kinase, which catalyzes the phosphorylationof 5-hydroxymethyldeoxycytidylate, dTMP, and dGMP, has been investigated by chemical modification of the protein
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
The chemical modification experiments presented in this study have provided further evidence for the existence of one catalytic site and have identified two regions of the primary sequence that are important for activity
Summary
The mechanismunderlying the unusual specificity of bacteriophage T4 deoxynucleotide kinase, which catalyzes the phosphorylationof 5-hydroxymethyldeoxycytidylate, dTMP, and dGMP, has been investigated by chemical modification of the protein. 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 identify two distant regions of the primary sequence that are likely to be closely associated in the active region of the folded protein. Whereas the recognition of three deoxynucleotides by one enzyme is interesting initself, thekineticexperiments of Bello and Bessman [1]indicate that thedeoxynucleotide substrates are competitive inhibitors, suggesting that one active site is responsible for all three activities. Enzyme was purified as previously described [12], with the additioonf a final Sephadex G-25 gel filtration step toremove residual ATP completely
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