title: Properties of bacteriophage T4 thymidylate synthase following mutagenic changes in the active site and folate binding region.
abstract: Amino acid replacements have been introduced in specific sites of bacteriophage T4 thymidylate synthase (T4-TS) to assess the role that these changes have on enzyme activity. Each of the conserved amino acids in the active-site region of T4-TS was modified, and the effects that these changes had on the kinetic and physical properties of this enzyme were measured. The mutations introduced were Pro-155-Ala (P155A), Cys-156-Ser (C156S), and His-157-Val (H157V) with the resulting synthases possessing kcat's of 10.3, 0.008, and 2.70 s-1, respectively, relative to that of the wild-type enzyme of 11.8 s-1. Equilibrium dialysis was performed on the wild-type and mutant enzymes to determine the binding constants for 2'-deoxyuridylate and 5-fluoro-2'-deoxyuridylate, and while in most cases the extent of binding of these nucleotides to the mutant proteins was reduced when compared with wild-type TS, the number of binding sites involved remained about 1 or less for the binary complex and almost 2 for the ternary complex. Heat and urea stability studies revealed that the mutant with the highest enzyme activity, P155A, was the most unstable, while spectrofluorometric analyses revealed that the structures of P155A and H157V were perturbed relative to the C156S and wild-type TSs. These studies are in agreement with others implicating the phylogenetically conserved active-site cysteine as playing an essential mechanistic role in the catalytic process promoted by TS. The proximal amino acids on either side of this cysteine, although also highly conserved, do not appear to affect the catalytic mechanism directly, but may do so indirectly through their influence on the conformation at the active site as well as other regions of the enzyme. Amino acids replacements were introduced also into the folate and deoxynucleotide 5'-phosphate binding sites of the T4-phage TS to ascertain the potential role that these amino acids play in the catalytic process. These positions were selected on the basis of previous chemical modification and X-ray crystallographic studies on Lactobacillus casei TS. Amino acid residues 48 and 49, which are in the putative folate binding site, were converted from lysines to arginines; in the former case, the mutated enzyme had less than 7% of the wild-type activity while in the latter, the mutated enzyme still retained about 60% of its activity.(ABSTRACT TRUNCATED AT 400 WORDS)
authors: Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201-0509.
authors: L
authors: L
authors: LaPat-Polasko
authors: G F
authors: GF
authors: Maley
authors: F
authors: F
authors: Maley
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005492:Folic Acid / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D012689:Sequence Homology, Nucleic Acid; D013050:Spectrometry, Fluorescence; D013604:T-Phages / Q000201:enzymology* / Q000235:genetics; D013940:Thymidylate Synthase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D015345:Oligonucleotide Probes; D005492:Folic Acid; D013940:Thymidylate Synthase