T4 endonuclease V [endodeoxyribonucease (pyrimidine dimer); deoxyribonuclease (pyrimikine dimer), EC 3.1.25.1] initiates repafr of damaed DNA by hydrolysis of the N-glycosyl bond at the 5' side of a pyrimidine photodimer in double-stranded DNA. To study one of the active sites of T4 endonudease V, systematic site-directed mutagenesis was performed on the synthetic T4 endonuclease V gene, in paraflel with three-dimensional structure analysis by x-ray crystallography. The mutant proteins were evaluated for DNA glycosylase activity using an oligonucleotide duplex (14-mer) containing a single thymidine dimer as a substrate. Replacement of either Glu-23 with glutamine or asparatic acid or Arg-3 with glutami completely abolished DNA glycosylase activity. Mutation of Arg-3 to lysine or of Arg-26 to glu or lysine in a basic amino acid cluster caused serious defects in DNA glycosylase activity, which are reflected in the increases in K. and decreases in kt of DNA glycosylase activity. On the other hand, substitutions of lysine for Arg-22 or of glutamine for Arg-117 or Lys-121 resulted in increases in the Km value. The completely inactive mutant proteins, E23Q and R3Q, in which glutamie was substituted for Glu-23 and Arg-3, respectively, were further investigated by CD spectroscopy for their ability to bind the oligonucleotide substrate. It was found that the E23Q protein retained specific substrate-binding ability, whereas the R3Q protein did not. These results indicate that Glu-23 plays an important role in catalysis of the DNA glycosylase reaction, and that Arg-3 is a crucial residue for substrate binding. In addition, Arg-22, Arg-26, Arg-117, and Lys-121 in the basic amino acid cluster also participate in substrate binding. We conclude that the basic amino acid cluster in T4 endonuclease V is an essential structure for DNA glycosylase activity.Endonuclease V [endo V; endodeoxyribonuclease (pyrimidine dimer); deoxyribonuclease (pyrimidine dimer), EC 3.1.25.1], found in bacteriophage T4, is responsible for excision repair of damaged DNA. The enzyme possesses two activities: a pyrimidine dimer DNA glycosylase (PD glycosylase) and an apyrimidinic/apurinic (AP) endonuclease ( Fig. 1) (1, 2). The gene (T4 denV) encoding T4 endo V was identified in association with the UV resistance of T4 phage and was shown to encode 138 amino acids (Fig. 2) (3). The protein recognizes a pyrimidine photodimer structure in a double-stranded DNA, hydrolyzes the N-glycosyl bond at the 5' side of a pyrimidine dimer, and cleaves the apyrimidinic phosphodiester bond via a (3-elimination reaction. In addition, T4 endo V is capable of binding and scanning nontarget DNA processively (4). The fact that such a small protein possesses two catalytic activities has engendered considerable interest in terms of the relationship between the two activities and the domain structure. As yet, no catalytic mechanism of any PD glycosylase has been elucidated.The aromatic residue-rich region, Trp-Tyr-Lys-Tyr-Tyr (residues 128-132), near the C terminus of ...

Prodrug activation via antibodies was examined by using the antibiotic chloramphenicol as a model drug. Based on the conformational change between substrate and product, this antibody-catalyzed reaction was designed to prevent product inhibition, thus enhancing turnover. Antibodies elicited against a phosphonate transition-state analogue were found to catalyze hydrolysis of a nonbioactive chloramphenicol monoester as a prodrug at a significantly higher rate above the uncatalyzed background reaction to regenerate chloramphenicol as a parent molecule. The antibody-catalyzed prodrug activation was tested by the paper-disc diffusion method using Bacillus subtilis as an indicator strain. The antibody 6D9 catalyzes the reaction with multiple turnover to generate enough chloramphenicol to inhibit bacterial growth, as indicated by a clear inhibitory zone after incubation with monoester. Using the same method, no inhibition was detected by incubation of either the monoester or the antibody alone. This result reveals that only the antibody hydrolytically activates the monoester, which can be expected to be a suitable prodrug, as it is resistant to the action of bacterial hydrolytic enzymes. The approach in this study demonstrates the use of catalytic antibody technology in medicine and may be applicable to drugs with undesirable effects, particularly in the field of cancer therapy.

T4 endonuclease V catalyzes the hydrolysis of the glycosyl bond of a thymine dimer in a DNA duplex and the cleavage of the 3'-phosphate by beta-elimination. We have previously identified a catalytic site for the first reaction (pyrimidine dimer-glycosylase activity) by systematic mutagenesis (Doi et al. Proc. Natl. Acad. Sci. USA 1992 in press) and by x-ray crystallography (Morikawa et al. Science, 256: 523-526, 1992). The results showed that replacement of Glu23 with either glutamine or aspartic acid completely abolished the glycosylase activity. We describe the investigation of the second reaction (apurinic/apyrimidinic endonuclease activity), using twenty two mutants of T4 endonuclease V plus a DNA mini duplex containing an abasic site. Replacement of Glu23 by glutamine abolished the second reaction, but replacement with aspartic acid did not. The pH optima of the mutant (23 Asp) and the wild type were found to be 5.0 and 5.5, respectively. We conclude that the carboxylate anion in position 23 may act as a general base in the beta-elimination reaction of the endonuclease.