| dc.description.abstract | (i) SHMT catalyzes the conversion of serine to glycine and the transfer of the hydroxymethyl group to H -folate to yield 5,10-CH -H -folate. This product serves as a carrier of one carbon units for the biosynthesis of a number of biologically important products such as thymidylate, purines, methionine, etc. (Fig. 2).
(ii) It was postulated earlier from our laboratory that SHMT was a regulatory enzyme and its activity was modulated by protein–ligand and protein–protein interactions (9, 23, 62).
(iii) These regulatory properties were abolished in rapidly proliferating tissues such as fetal liver and neoplastic tissues (61, 62).
(iv) The literature on the structure, function and regulation of SHMT is selectively reviewed.
(v) SHMT was earlier purified to homogeneity from monkey, sheep and human livers, and from mung bean seedlings, and all these enzyme preparations exhibited homotropic interactions with H -folate. Positive heterotropic effects were observed with BDB, whereas negative heterotropic interactions were exhibited with glycine (9, 23, 62, 26).
(vi) A re-examination of the presence of allosteric effects with the enzyme revealed that this was not an artefact but an inherent property of the enzyme (23).
(vii) It was demonstrated that this regulation of the enzyme was absent in extracts of neoplastic tissues. A proteinaceous factor present in these extracts altered the cooperative interactions (62).
(viii) It was also shown by other workers that serine metabolism was channelled towards its biosynthesis by increased activities of the enzymes forming the amino acid, and the enzymes involved in degradation were selectively repressed (65).
(ix) All these observations clearly indicated that SHMT was an important enzyme in providing increased amounts of precursors for DNA biosynthesis in proliferating tissues, and this led to the postulate that it could act as an alternate target for chemotherapy.
(x) Mechanism based inactivators are very useful chemotherapeutic agents in that the inactivation is highly specific and expected to have minimal toxicity. PLP dependent enzymes have been extensively studied as targets for mechanism based inactivators and since SHMT is a PLP containing protein, the literature on mechanism based inactivators is reviewed.
(xi) The objectives of the present investigation were:
a) Standardization of a convenient procedure for obtaining larger amounts of SHMT needed for the physico chemical studies involved in examining the interactions of inhibitors at the active site;
b) Examination of the inhibition of SHMT by folate analogs such as 5,8 dideazafolate derivatives, and serine analogs such as substituted alanines and aminooxy compounds; and
c) Elucidation of the mechanism of interaction of serine analogs such as O amino D serine, aminooxyacetate, L canaline, hydroxylamine and other analogs of serine. The mode of interaction of aminooxy compounds was examined to test the hypothesis that O substituted hydroxylamine derivatives of substrate (serine or glycine) would yield active site directed reagents which inhibit SHMT very potently and specifically.
(xii) The materials and methods used in this study are described. These include estimation of enzyme activity, spectral measurements including CD, fluorescence and stopped flow spectrophotometry, electrophoretic methods, antibody preparation, HPLC, etc.
(xiii) Starting from 1 kg of sheep liver and using ion exchange chromatography on CM Sephadex, ammonium sulfate fractionation and gel filtration on Sephacryl S 200, 200 mg of enzyme protein was obtained (Table 9).
(xiv) The purity of the enzyme preparation was established by PAGE (Fig. 42), SDS PAGE (Fig. 43) and an immunodouble diffusion test (Fig. 44).
(xv) This enzyme preparation had a specific activity of 7.2 (µmol of HCHO/min/mg protein) (Table 9) compared to 6.0 units reported earlier (23).
(xvi) The enzyme obtained by this procedure was identical in its kinetic (Figs. 46 & 47) and physico chemical (Figs. 48–54) properties with that reported earlier (23). This demonstrated that the procedure yielded a pure enzyme preparation suitable for the studies reported in this thesis.
(xvii) The inhibition of SHMT activity by about 30 compounds structurally related to H -folate (Figs. 55–58) was examined. None of these compounds inhibited SHMT activity very effectively (Table 10).
(xviii) Substituted alanines (Fig. 59), which function as mechanism based inactivators of many PLP dependent enzymes such as AspAT (120–122) and alanine racemase (138–141), also inhibited SHMT activity (Table 11). However, like the folate analogs, these compounds were not good inhibitors of SHMT.
(xix) A preliminary examination of the inhibition of SHMT by aminooxy compounds (Fig. 60) indicated that these compounds were potent inhibitors of the enzyme (Table 12) and the mechanism of inhibition was further studied.
(xx) Earlier observations from this laboratory (242) indicated that DCS, a mechanism based inhibitor of alanine racemase (244, 245) and AspAT (251) and also an antitumor compound (241), inhibited sheep liver SHMT by forming a Schiff base with PLP which dissociated from the active site leaving behind an inactive apoenzyme.
(xxi) Hydroxylamine is a well known inhibitor of PLP dependent enzymes and it was postulated that substrate related hydroxylamine derivatives could be effective inhibitors of SHMT.
(xxii) OADS was a reversible non competitive inhibitor (K = 1.8 µM), when serine was the varied substrate (Figs. 72 & 73). The inhibition was completely reversed by PLP (Table 14).
(xxiii) The first step in the interaction of OADS with the enzyme was the disruption of the internal Schiff base (Fig. 74) and was characterized by the rapid disappearance of the absorbance at 425 nm (Fig. 82) and CD intensity at 430 nm (Fig. 76). Concomitantly, there was a rapid increase in absorbance (Fig. 82) and CD intensity at 390 nm (Fig. 76). These spectral properties enabled identification of this intermediate as PLP.
(xxiv) The changes in absorbance at 425 and 388 nm were too rapid for kinetic analysis by conventional spectroscopy. Stopped flow studies enabled evaluation of the rate constant at 425 and 388 nm to be 6.3 × 10 M ¹ s ¹ (Figs. 83 & 84).
(xxv) Time difference spectra (Fig. 86), constructed by measuring rapid absorbance changes at different wavelengths (350–460 nm) (Fig. 85), revealed that no additional spectrally absorbing intermediates were present before PLP formation.
(xxvi) These changes were followed by a slow unimolecular step (k = 2 × 10 ³ s ¹) leading to the formation of PLP–OADS oxime (Fig. 90).
(xxvii) Formation of PLP–OADS oxime was confirmed by its absorbance (Fig. 79), fluorescence spectrum (Fig. 80A) and HPLC retention time (Fig. 80C).
(xxviii) The PLP–OADS oxime was displaced from the enzyme by the addition of PLP, as evidenced by complete restoration of enzyme activity (Table 14) and regain of spectral properties of the holoenzyme (Figs. 76 & 77).
(xxix) A minimal kinetic scheme (Fig. 96) was proposed for the interaction of OADS with sheep liver SHMT, and the unique feature of the mechanism was the formation of PLP as an intermediate.
(xxx) Aminooxy compounds such as AAA and hydroxylamine inhibited very potently (IC = 2–3 µM) (Table 18), whereas L canaline inhibited very poorly (~100 µM).
(xxxi) The mode of interaction of AAA with SHMT, monitored by changes in the absorption spectrum (Fig. 105), indicated that the reaction was similar to OADS.
(xxxii) The disappearance of absorbance at 425 nm on interaction of AAA with SHMT was biphasic (Fig. 106), with rate constants of 191 and 19 s ¹.
(xxxiii) The formation of PLP–AAA oxime, monitored by the disappearance of the 388 nm absorbance (Fig. 107), showed concentration dependency with a second order rate constant of 5.2 × 10 M ¹ s ¹. This indicated that AAA interacted differently with SHMT.
(xxxiv) L Canaline, a higher homolog of OADS, was not an effective inhibitor, and formation of PLP as an intermediate was not clearly detected (Fig. 101).
(xxxv) Hydroxylamine reacted rapidly with the enzyme to form the PLP oxime without any detectable intermediate (Fig. 102).
(xxxvi) Interaction of hydroxylamine with the enzyme showed a biphasic first order plot (Figs. 112 & 113). The first step showed concentration dependency with a rate constant of 2.1 × 10 M ¹ s ¹, whereas the second step did not show any concentration effect and had a rate constant of 0.8 s ¹ (at 1 mM hydroxylamine).
(xxxvii) The reaction of DCS with the enzyme (Fig. 117) was slower (1.6 × 10 ³ s ¹) than with the aminooxy compounds.
(xxxviii) These observations indicated that aminooxy compounds (Fig. 120), which are structural analogs of serine (OADS, AAA), formed PLP as an intermediate prior to oxime formation, whereas with hydroxylamine such an intermediate could not be detected. OADS and AAA were effective inhibitors (2–3 µM), whereas L canaline, DCS, DL 2,3 diaminopropionic acid and DL 2,4 diaminobutyric acid were poor inhibitors.
(xxxix) In conclusion, the results and discussion presented in this thesis suggested that aminooxy compounds related to the substrate (OADS/AAA) can effectively and specifically inhibit SHMT, and this class of compounds can probably be developed as cancer chemotherapeutic agents. | |
