Methylenetetrahydrofolate reductase from Escherichia coli: elucidation of the kinetic mechanism by steady-state and rapid-reaction studies.

The flavoprotein methylenetetrahydrofolate reductase (MTHFR) from Escherichia coli catalyzes the reduction of 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate) to 5-methyltetrahydrofolate (CH(3)-H(4)folate) using NADH as the source of reducing equivalents. The enzyme also catalyzes the transfer of reducing equivalents from NADH or CH(3)-H(4)folate to menadione, an artificial electron acceptor. Here, we have determined the midpoint potential of the enzyme-bound flavin to be -237 mV. We have examined the individual reductive and oxidative half-reactions constituting the enzyme's activities. In an anaerobic stopped-flow spectrophotometer, we have measured the rate constants of flavin reduction and oxidation occurring in each half-reaction and have compared these with the observed catalytic turnover numbers measured under steady-state conditions. We have shown that, in all cases, the half-reactions proceed at rates sufficiently fast to account for overall turnover, establishing that the enzyme is kinetically competent to catalyze these oxidoreductions by a ping-pong Bi-Bi mechanism. Reoxidation of the reduced flavin by CH(2)-H(4)folate is substantially rate limiting in the physiological NADH-CH(2)-H(4)folate oxidoreductase reaction. In the NADH-menadione oxidoreductase reaction, the reduction of the flavin by NADH is rate limiting as is the reduction of flavin by CH(3)-H(4)folate in the CH(3)-H(4)folate-menadione oxidoreductase reaction. We conclude that studies of individual half-reactions catalyzed by E. coli MTHFR may be used to probe mechanistic questions relevant to the overall oxidoreductase reactions.

Folate activation and catalysis in methylenetetrahydrofolate reductase from Escherichia coli: roles for aspartate 120 and glutamate 28.

The flavoprotein Escherichia coli methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate) to 5-methyltetrahydrofolate (CH(3)-H(4)folate). The X-ray crystal structure of the enzyme has revealed the amino acids at the flavin active site that are likely to be relevant to catalysis. Here, we have focused on two conserved residues, Asp 120 and Glu 28. The presence of an acidic residue (Asp 120) near the N1-C2=O position of the flavin distinguishes MTHFR from all other known flavin oxidoreductases and suggests an important function for this residue in modulating the flavin reactivity. Modeling of the CH(3)-H(4)folate product into the enzyme active site also suggests roles for Asp 120 in binding of folate and in electrostatic stabilization of the putative 5-iminium cation intermediate during catalysis. In the NADH-menadione oxidoreductase assay and in the isolated reductive half-reaction, the Asp120Asn mutant enzyme is reduced by NADH 30% more rapidly than the wild-type enzyme, which is consistent with a measured increase in the flavin midpoint potential. Compared to the wild-type enzyme, the mutant showed 150-fold decreased activity in the physiological NADH-CH(2)-H(4)folate oxidoreductase reaction and in the oxidative half-reaction involving CH(2)-H(4)folate, but the apparent K(d) for CH(2)-H(4)folate was relatively unchanged. Our results support a role for Asp 120 in catalysis of folate reduction and perhaps in stabilization of the 5-iminium cation. By analogy to thymidylate synthase, which also uses CH(2)-H(4)folate as a substrate, Glu 28 may serve directly or via water as a general acid catalyst to aid in 5-iminium cation formation. Consistent with this role, the Glu28Gln mutant was unable to catalyze the reduction of CH(2)-H(4)folate and was inactive in the physiological oxidoreductase reaction. The mutant enzyme was able to bind CH(3)-H(4)folate, but reduction of the FAD cofactor was not observed. In the NADH-menadione oxidoreductase assay, the mutant demonstrated a 240-fold decrease in activity.

Purification and properties of NADH-dependent 5, 10-methylenetetrahydrofolate reductase (MetF) from Escherichia coli.

A K-12 strain of Escherichia coli that overproduces methylenetetrahydrofolate reductase (MetF) has been constructed, and the enzyme has been purified to apparent homogeneity. A plasmid specifying MetF with six histidine residues added to the C terminus has been used to purify histidine-tagged MetF to homogeneity in a single step by affinity chromatography on nickel-agarose, yielding a preparation with specific activity comparable to that of the unmodified enzyme. The native protein comprises four identical 33-kDa subunits, each of which contains a molecule of noncovalently bound flavin adenine dinucleotide (FAD). No additional cofactors or metals have been detected. The purified enzyme catalyzes the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, using NADH as the reductant. Kinetic parameters have been determined at 15 degreesC and pH 7.2 in a stopped-flow spectrophotometer; the Km for NADH is 13 microM, the Km for CH2-H4folate is 0.8 microM, and the turnover number under Vmax conditions estimated for the reaction is 1,800 mol of NADH oxidized min-1 (mol of enzyme-bound FAD)-1. NADPH also serves as a reductant, but exhibits a much higher Km. MetF also catalyzes the oxidation of methyltetrahydrofolate to methylenetetrahydrofolate in the presence of menadione, which serves as an electron acceptor. The properties of MetF from E. coli differ from those of the ferredoxin-dependent methylenetetrahydrofolate reductase isolated from the homoacetogen Clostridium formicoaceticum and more closely resemble those of the NADH-dependent enzyme from Peptostreptococcus productus and the NADPH-dependent enzymes from eukaryotes.

Cisplatin.

Cisplatin is a widely used anti-cancer drug that is exceptionally effective against testicular cancer. trans-DDP, the geometric isomer of cisplatin, is ineffective as a chemotherapeutic agent. The anti-tumour activity of cisplatin is generally attributed to its formation of DNA adducts, both intrastrand and interstrand crosslinks, which induce structural distortions in DNA. The DNA adducts of cisplatin are thought to mediate its cytotoxic effects by inhibiting DNA replication and transcription and, ultimately, by inducing programmed cell death, or apoptosis. The adducts of both cis- and trans-DDP are removed from DNA by the nucleotide-excision-repair pathway. Cellular proteins possessing certain DNA-binding motifs, including the HMG domain, bind selectively to DNA modified by cisplatin, but not to DNA adducts of trans-DDP; evidence suggests a possible role for these proteins in modulating cisplatin cytotoxicity. Both intrinsic and drug-induced resistance often limit the success of cisplatin; several specific mechanisms of cisplatin resistance have been identified.

Human testis-determining factor SRY binds to the major DNA adduct of cisplatin and a putative target sequence with comparable affinities.

cis-Diamminedichloroplatinum(II) (cis-DDP or cisplatin) is a widely used anticancer drug that is most effective against tumors of the testis. Although cisplatin is believed to mediate its cytotoxicity through the formation of DNA adducts, the precise biochemical mechanisms underlying its antitumor activity and selectivity for testicular tumors remain elusive. Of significance are the high-mobility group (HMG) domain and other proteins that bind specifically to cisplatin-DNA adducts. The present study focuses on the testis-specific HMG domain protein human SRY (hSRY). The full-length hSRY protein and its HMG domain region alone were expressed in Escherichia coli and purified to homogeneity. The affinities and specificities of full-length hSRY and the hSRY-HMG domain for 20 bp DNAs containing a single cis-[Pt(NH3)2{d(GpG)-N7(1), -N7(2)}] intrastrand cross-link or a putative hSRY target site in the CD3epsilon gene enhancer (AACAAAG) were determined in electrophoretic mobility shift assays. Full-length hSRY bound to the major 1,2-d(GpG) cisplatin adduct with a Kd(app) of 120 +/- 10 nM and exhibited a 20-fold specificity over unmodified DNA. The HMG domain of hSRY was sufficient for this interaction. The hSRY-HMG domain recognized the 1,2-d(GpG) intrastrand cross-link with higher affinity [Kd(app) = 4 +/- 0.7 nM] but with lower specificity (5-fold) than the full-length protein. The affinities of full-length hSRY and the hSRY-HMG domain for a single cisplatin-DNA adduct were comparable to those for the putative target sequence AACAAAG. These data suggest that cisplatin-DNA adducts may compete with specific DNA sequences in vivo for the binding of human SRY. A possible role for this testis-specific protein in the cytotoxicity and organotropic specificity of cisplatin for testicular tumors is proposed.