Isolation, kinetic mechanism and active site studies on sheep liver 5, 10-methylenetetrahydrofolate reductase

Abstract

(i) A major landmark in the history of science is the discovery of the biochemical and physiological functions of vitamins. Folic acid, which was isolated from spinach leaves for the first time, was shown to be an anti anaemia factor. This vitamin, in its coenzyme forms (Fig. 1), functions as a carrier of one carbon fragments required for the biosynthesis of methionine, thymidylate, purines, tRNA and a large number of end products that arise by the transfer of methyl groups from S adenosylmethionine (Fig. 2). (ii) The pathway for the interconversion of folate coenzymes involves several oxidoreductases such as dihydrofolate reductase, thymidylate synthase, 5,10 methylene tetrahydrofolate dehydrogenase and 5,10 CH H folate reductase, as well as serine hydroxymethyltransferase (SHMT), which channels C fragments from serine into the pathway. (iii) As this thesis pertains to the study of 5,10 CH H folate reductase, a survey of the literature on DHFR, TS, 5,10 CH H folate dehydrogenase and 5,10 CH H folate reductase is presented. DHFR has attracted considerable attention as a target for chemotherapy against cancer. In spite of extensive investigations, the hope of discovering drugs better than MTX or aminopterin has not been completely fulfilled. Although extensive and elegant work by Schimke and others has indicated gene amplification as a mode of drug resistance, other possibilities such as impaired transport, and the presence of multiple and altered forms of the enzyme have gained prominence in recent years. (iv) TS, like DHFR, has also been considered as a possible site for drug action. Detailed studies on its mechanism have provided insight into how antitumor drugs inhibit DNA synthesis and thereby cause tumor regression. (v) 5,10 CH H folate dehydrogenase is a good example of a single polypeptide chain catalyzing multiple catalytic functions. This phenomenon, however, is not universal among enzymes isolated from different sources, suggesting the possible existence of alternative mechanisms of regulation. (vi) 5,10 CH H folate reductase had resisted several attempts at purification until recently. Matthews and her collaborators, in a series of papers, have reported the isolation of this enzyme from pig liver in pure form, along with identification and characterization of the intermediates involved in the enzymatic reduction of 5,10 CH H folate. (vii) The aim and scope of the present investigation are as follows. Although extensive studies have been carried out on these enzymes, very little information is available on regulation of the pathway by allosteric interactions or by protein-protein interactions among related enzymes. This laboratory has carried out extensive investigations on allosteric regulation of SHMT in normal and neoplastic tissues. As part of the study on regulation of folate metabolism, it was of interest to purify and characterize 5,10 CH H folate reductase, the next enzyme in the pathway. Earlier observations on the interaction of Cibacron Blue with SHMT prompted attempts to use this dye as an affinity ligand to purify the enzyme. This thesis reports a method for purification of sheep liver 5,10 CH H folate reductase, establishes the kinetic mechanism of the reaction, describes its interaction with Cibacron Blue and Procion Red HE 3B (two pseudo affinity ligands), and identifies amino acids at the active site of the enzyme. (viii) The procedures employed include assay of 5,10 CH H folate reductase activity, SDS PAGE, determination of molecular weight of the native enzyme and its subunits, absorbance measurements, difference spectral studies, fluorescence measurements and circular dichroism studies. (ix) The reductase from sheep liver was purified by (NH ) SO fractionation, acid precipitation, DEAE Sephacel ion exchange chromatography and Blue Sepharose affinity chromatography (Table 3). (x) Homogeneity of the enzyme was evident from a single band on SDS PAGE (Fig. 6A), a single symmetrical peak on analytical ultracentrifugation (Fig. 6B), gel filtration, and a single precipitin line on Ouchterlony double diffusion (Fig. 6C). (xi) The enzyme was a homodimer with molecular weight 166,000 ± 5,000 (Fig. 7). The enzyme was stable for two weeks in the presence of 10% glycerol. During purification, FAD was lost and external addition of FAD restored activity. (xii) The enzyme, a flavoprotein, functioned optimally at pH 6.5 and 37°C (Fig. 8). The enzyme exhibited (a) 5 CH H folate-menadione oxidoreductase, (b) NADPH-menadione oxidoreductase, and (c) NADPH-5,10 CH H folate oxidoreductase activities. The specific activity varied from 1-1.3 mol/min/mg protein. The saturation with 5 CH H folate was hyperbolic with Km = 132 M (Fig. 9). In NADPH-menadione oxidoreductase assay, Km values were 16 M for NADPH and 2.5 M for menadione (Fig. 10). (xiii) When menadione was varied at different fixed NADPH concentrations, parallel lines were obtained (Fig. 15). Similarly, varying NADPH at fixed menadione concentrations gave parallel lines (Fig. 16). (xiv) When NADPH was varied at fixed NADP and unsaturating menadione concentrations, non competitive inhibition was observed (Fig. 17). At saturating menadione, NADP showed no inhibition (Fig. 18). Competitive inhibition was observed when menadione was varied at unsaturating NADPH and different fixed NADP concentrations (Fig. 19). (xv) These double reciprocal patterns in initial velocity and product inhibition studies matched predicted patterns for a ping pong mechanism (Fig. 21). (xvi) By analogy with the mechanism proposed for NADPH-menadione oxidoreductase, a similar kinetic mechanism is suggested for reduction of 5,10 CH H folate to 5 CH H folate. (xvii) A feature of this mechanism was that both NADPH and 5 CH H folate interacted with the same form of the enzyme. Thus, inhibition by Cibacron Blue, which interacts with both NADPH and folate binding domains, was examined. (xviii) The dye completely inhibited the enzyme (Fig. 25), and dilution reversed inhibition. Steady state kinetic analysis showed competitive inhibition with respect to 5 CH H folate (Fig. 26; Ki = 1.2 M) and competitive inhibition with NADPH (Fig. 27; Ki = 0.5 M). This suggests binding at the active site. (xix) Dye difference spectra showed an absorbance maximum at 690 nm and a trough at 600 nm (Fig. 30). Absorbance at 690 nm increased with dye concentration (Fig. 31), giving Kd = 6 M. Addition of 5 CH H folate or NADPH reversed spectral changes (Fig. 32). KCl reversal suggested electrostatic contributions. (xx) Fluorescence quenching also yielded Kd 1 M and indicated a single binding site (Fig. 35). (xxi) These results and the red shift (Fig. 30) suggest that the dye first binds to a hydrophobic pocket and is stabilized by electrostatic forces. (xxii) Procion Red, a structurally similar dye, also competitively inhibited the enzyme (Fig. 29; Ki = 1 M), consistent with fluorescence quenching results (Fig. 37). Similar Ki and Kd values indicate similar interactions. (xxiii) Electrostatic interactions and negatively charged substrate groups suggested involvement of positively charged residues (arginine, histidine). Hydrophobic dye interactions and known active site tryptophans in dehydrogenases suggested tryptophan involvement. Charge transfer with FAD is also possible. Chemical modification studies were therefore employed. (xxiv) Inactivation by phenylglyoxal followed pseudo first order kinetics (Fig. 42), giving a second order rate constant of 0.05 M ¹ min ¹. Stoichiometry indicated reaction with two arginine residues. Protection by 5 CH H folate (Fig. 43) indicated interaction at the substrate site. (xxv) DEPC modification (Fig. 44) implicated histidine. Protection by niacin and NADPH (Fig. 45) suggested involvement at the NADPH binding site. Reversal by hydroxylamine (Table 10) and absorption at 235 nm (Fig. 46) confirmed histidine modification. (xxvi) Tryptophan involvement was suggested by inactivation with NBS (Fig. 48) and protection by folate (Fig. 49). A second order rate constant of 500 M ¹ min ¹ was obtained. (xxvii) A plausible model is proposed: - 5,10 CH H folate binds in a hydrophobic pocket containing a tryptophan residue and interacts electrostatically via its glutamate carboxyl groups with enzyme arginine residues. - Tryptophan likely participates in a charge transfer complex with FAD. - NADPH interacts with histidine residues. These interactions stabilize substrate binding and facilitate electron transfer, enabling product formation and release.