The site for the allosteric activator GTP of Escherichia coli UMP kinase

UMP kinase (UMPK), a key bacterial pyrimidine nucleotide biosynthesis enzyme, is UTP-inhibited and GTP-activated. We delineate the GTP site of Escherichia coli UMPK by alanine mutagenesis of R92, H96, R103, W119 or R130, abolishing GTP activation; of S124 and R127, decreasing affinity for GTP; and of N111 and D115, with little detrimental effect. We exclude the correspondence with the modulatory ATP site of Bacillus anthracis UMPK, confirming the functionality of the GTP site found by Evrin. Mutants R92A, H96A and R127A are constitutively activated, suggesting key roles of these residues in allosteric signal transduction and of positive charge neutralization in triggering activation. No mutation hampered UTP inhibition, excluding overlapping of the UTP and GTP sites.