Substrate Specificity and Radioactive Labeling Studies Establish that the Histidine Triad Nucleotide Binding Proteins (Hints) are Nucleoside Phosphoramidases and Protein Nucleotidylases

Human and E. coli histidine triad nucleotide‐binding proteins (Hints) have been established as purine nucleoside phosphoramidases. To study the mechanism of this of hydrolysis, a continuous fluorescence based assay was developed. Both the human and bacterial enzymes preferred purines over pyrimidines. However, while a stereochemical preference was not observed for E. coli hinT, human Hint1 (hHint1) preferred D‐tryptophan substrates. The biochemical function of the phosphoramidase activity has remained obscure. It has been proposed that certain post‐translationally nucleotidylated proteins may be natural substrates of Hints. To address this hypothesis E. coli cell lysates were incubated with [α‐ 32 P]‐ATP and [α‐ 32 P]‐GTP to generate adenylylated and guanylylated proteins. Proteins with M.W. of 30 kDa and 59 kDa appeared to be the most intensively guanylylated bands. Post‐labeling incubations with either E. coli hinT or hHint1 resulted in loss of the labeling of the 59 kDa band. Unexpectedly, E. coli cell lysates and of the human cancer cell extracts were shown to label E. coli hinT when incubated with [α‐ 32 P]‐ATP. In contrast, hHint1 was labeled with either [α‐ 32 P]‐ATP, [α‐ 32 P]‐GTP, [γ‐ 32 P]‐ATP, [γ‐ 32 P]‐GTP, or [ 32 P]‐cGMP in MDA‐MB‐468 cells extracts. Our results demonstrate that Hints are protein nucleotidylases and post‐translatioally modified by nucleotidyltransferases.