Structural Properties of UMP-Kinase from Escherichia coli:  Modulation of Protein Solubility by pH and UTP

Abstract

UMP-kinase from Escherichia coli, unlike the analogous enzyme from eukaryotic organisms, is an oligomeric protein subjected to complex regulatory mechanisms in which UTP and GTP act as allosteric effectors. While the enzyme has an unusually low solubility at neutral pH (< or = 0.1 mg of protein/ mL), its solubility increases markedly above pH 8 and below pH 4. Furthermore, the solubility of the bacterial UMP-kinase at neutral pH is greatly enhanced in the presence of Mg-free UTP. Thermal denaturation experiments have demonstrated that UTP also increases the stability of the protein. Fourier-transform infrared spectroscopy and circular dichroism show that the secondary structure of the protein is the same at neutral and at alkaline pH. These data indicate that variations in enzyme solubility must be related to subtle changes in the tertiary and/or quaternary structure which modulate the exposure of hydrophobic surfaces in the protein molecule. A variant of UMP-kinase, obtained by site-directed mutagenesis (Asp159Asn), which is similar to the wild-type enzyme in its stability and kinetic properties, has a much increased water solubility (> 5 mg protein/mL) even at neutral pH. This suggests that salt bridges may be involved in the equilibrium between the soluble and aggregated forms of the wild-type enzyme, and that conformational changes induced upon binding of UTP increase the protein solubility by disrupting these salt bridges.

UMP kinase fromStreptococcus pneumoniae: evidence for co-operative ATP binding and allosteric regulation

“…The binding of UTP may induce the protein to adopt a more extended conformation, where exposure of hydrophobic side chains would favour the formation of large polymers or aggregates. Changes in the solubility of the protein have also been described for UMP kinase from E. coli [15,16]. The effect of the substrate UMP on the conformation of S. pneumoniae enzyme is intriguing.…”

“…UMP would then induce the same conformational change as UTP and it would also probably act as an inhibitor. Serina et al [15] have described inhibition by excess of UMP with the E. coli UMP kinase, at pH 7.4 and not at pH 6.0. With the S. pneumoniae UMP kinase, no inhibition by excess of UMP was ever obtained either at pH 7.5 or 6.5.…”

“…In addition, molecular modelling studies predict that the E. coli UMP kinase structure shares similarities with carbamate kinases and acetylglutamate kinases [14]. To date, only UMP kinases from E. coli and B. subtilis have been overexpressed, purified and characterized [12,13,[15][16][17]. Both proteins are inhibited by UTP and activated by GTP.…”

Purine and Pyrimidine Nucleotide Synthesis and Metabolism

“…This enzyme has been studied from a variety of bacterial sources (Valentin-Hansen, 1978, Yamanaka et al, 1992, Serina et al, 1995, Serina et al, 1996. The bacterial enzyme is allosterically regulated by both GTP and UTP (Serina et al, 1995).…”

Structure–function relationships of UMP kinases from pyrH mutants of Gram-negative bacteria

“…First, UMP kinase is the only NMP kinase known to be endowed with a second biological role besides its catalytic activity, namely, the participation of UMP kinase in regulating the transcription of the carAB operon (Kholti et al, 1998). Second, UMP kinase is a homohexamer, and its catalytic activity is allosterically regulated by GTP/UTP (Serina et al, 1996). A third feature is that UMP kinase displays very low sequence similarity to other NMP kinases, which should allow for the design of highly specific inhibitors of its catalytic activity (Serina et al, 1995).…”