Protein-protein interactions involving T4 phage-coded deoxycytidylate deaminase and thymidylate synthase.
The enzymes deoxycytidylate deaminase (EC) and thymidylate synthase (EC) are functionally associated with one another, since they catalyze sequential reactions. In T4 coliphage infection the two enzymes are found in dNTP synthetase, a multienzyme complex for deoxyribonucleotide biosynthesis. Protein-protein interactions involving the phage-coded forms of these two enzymes have been explored in three experiments that use the respective purified protein as an affinity ligand. First, an extract of radiolabeled T4 proteins was passed through a column of immobilized enzyme (either dTMP synthase or dCMP deaminase), and the specifically bound proteins were identified. Second, two mutant form of dCMP deaminase (H90N and H94N), altered in presumed zinc-binding sites, were analyzed similarly, with the results suggesting that some, but not all, interactions require normal structure near the catalytic site. Third, affinity chromatography using either enzyme as the immobilized ligand, revealed interactions between the two purified enzymes in the absence of other proteins. In these experiments we noted a significant effect of dCTP, an allosteric modifier of dCMP deaminase, upon the interactions.
McGaughey
K M
KM
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331-7503, USA.
Wheeler
L J
LJ
Moore
J T
JT
Maley
G F
GF
Maley
F
F
Mathews
C K
CK
D017122:Bacteriophage T4 / Q000201:enzymology*; D003844:DCMP Deaminase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003845:Deoxycytosine Nucleotides / Q000494:pharmacology; D004800:Enzymes, Immobilized / Q000378:metabolism; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D013940:Thymidylate Synthase / Q000302:isolation & purification / Q000378:metabolism*
D003845:Deoxycytosine Nucleotides; D004800:Enzymes, Immobilized; D009097:Multienzyme Complexes; C024107:2'-deoxycytidine 5'-triphosphate; D013940:Thymidylate Synthase; D003844:DCMP Deaminase