Identification of a site necessary for allosteric regulation in T4-phage deoxycytidylate deaminase.
An allosteric inhibitor of dCMP deaminase, dTTP, forms a photolabile covalent bond with T4-phage dCMP deaminase in the presence of UV light at 254 nm. The importance of the methyl group in this process is supported by the findings that dUTP, also an allosteric inhibitor, does not photofix to the enzyme and that tritium is released from [methyl-3H dTTP during the course of the photofixation. That the bond formed is photolabile is demonstrated by the fact that tritium is released by about 10-fold over the amount of nucleotide that is photofixed. The amino acid that covalently binds dTTP in T4-dCMP deaminase was identified as Phe112. On conversion of Phe112 to an alanine by site-directed mutagenesis, there was a dramatic change in the enzyme's response to its allosteric effectors when measured early in the reaction, in that the mutant enzyme was as active as the wild-type even in the absence of dCTP and was only weakly inhibited by dTTP. However, after 10-15% of the substrate had been deaminated, the reaction rate fell off rather markedly, indicating either that an inhibitor was being accumulated on the enzyme or that the enzyme was being irreversibly inactivated with time. That the latter was not the case was shown by the addition of dCTP to the reaction, which restored the rate to that expected when it was present initially. Furthermore, we showed that, consistent with the observed loss of allosteric regulation by dCTP and dTTP, the affinity of the mutant enzyme for dTTP and dCTP as determined by binding studies was greatly reduced relative to the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Moore
J T
JT
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany.
Cieśla
J M
JM
Changchien
L M
LM
Maley
G F
GF
Maley
F
F
D000409:Alanine / Q000235:genetics; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000201:enzymology*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003844:DCMP Deaminase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral; D008969:Molecular Sequence Data; D009154:Mutation; D010449:Peptide Mapping; D010455:Peptides / Q000032:analysis; D010649:Phenylalanine / Q000235:genetics; D010777:Photochemistry; D013942:Thymine Nucleotides / Q000378:metabolism; D014357:Trypsin
D004279:DNA, Viral; D010455:Peptides; D013942:Thymine Nucleotides; D010649:Phenylalanine; D014357:Trypsin; D003844:DCMP Deaminase; D000409:Alanine; C024157:thymidine 5'-triphosphate