Abstract
CTP synthetase from Escherichia coli catalyzes exchange of 18O from the beta gamma-bridge position of [gamma-18O4] ATP into the beta-nonbridge position. This positional isotope exchange occurs in the presence of UTP and MgCl2 but in the absence of NH3. The enzyme also has an ATPase activity in the presence of UTP that occurs under conditions that are identical to those used in the positional isotope exchange experiments. These data provide evidence for the stepwise nature of the reactions catalyzed by CTP synthetase with the initial step involving phosphorylation of UTP by ATP. The relative rate of the isotope exchange reaction is approximately 3 times faster than the ATPase reaction, but the isotope exchange rate is approximately 3% of the overall rate in the presence of NH3. These results are consistent with the ATPase reaction involving attack of water on the phosphorylated intermediate (4-phospho-UTP). The positional isotope exchange reaction is independent of the UTP concentration above saturating levels of UTP demonstrating that the order of addition of substrates is UTP followed by ATP and then NH3.
Highlights
From the $Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802 and the §Departmentof Biochemistry, School of Medicine, University of Minnesota at Duluth,Duluth, Minnesota 55812
ATPase activity in the presence of UTP that occurs under conditions thaat re identical to those usedin the positional isotope exchange experiments
-8.4 ppm CTP synthetase was purified by the method of Anderson (1983)
Summary
From the $Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802 and the §Departmentof Biochemistry, School of Medicine, University of Minnesota at Duluth,Duluth, Minnesota 55812. Above was the reported lack of ATP-ADP exchange (51%of Vmm)both inthe presence and absence of UTP (Levitzki and Koshland, 1971). The positional isotope exchange (PIX1) techence of UTP, ATP, and M$+, the enzyme aggregates to a nique was developedby Midelfort and Rose (1976) tetramer (Anderson, 1983). As shown in Scheme 1,the reaction was thought to proceed enzymatic reactions that have intermediates as part of the (Koshland and Levitzki, 1974) by theattack of ammonia mechanism (DeBrosse and Villafranca, 1983). This method (alone or generated from glutamine) onthe 4-carbon atom of has proven useful in many laboratories for the study of several.
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