Assay method for antitumor l-methionine γ-lyase: comprehensive kinetic analysis of the complex reaction with l-methionine

The Role of Cysteine 116 in the Active Site of the Antitumor EnzymeL-Methionine γ-Lyase fromPseudomonas putida

“…The mutant forms of MGL Pp were made on wild-type construct pMGL1204, 27) using the QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA). The kit employs double-stranded DNA as a template, two complementary oligonucleotide primers containing a degenerate codon (NNN; N = A, T, G, or C) at position Cys116, which was constructed by Proligo Japan (Tokyo), as follows: sense primer, 5 0 -CCCTGTACG-GCNNNACCTTTGCCTTCCTGC-3 0 , antisense primer, 5 0 -GCAGGAAGGCAAAGGTNNNGCCGTACAGGG-3 0 , and Dpn I endonucleases to digest the parental DNA template.…”

Production of methionine γ- lyase in recombinant Citrobacter freundii bearing the hemoglobin gene

“…This activity was first found using dried cells of E. coli and Proteus vulgaris (4). MGL have been isolated, purified, and characterized from several microorganisms such as Pseudomonas putida, Clostridium sporogenes, Aeromonas sp., Citrobacter intermedius, Citrobacter freundii, Brevibacterium linens, Trichomonas vaginalis, and Porphyromonas gingivalis (3)(4)(5)(6)(7)(8)(9)(10). MGL has not been found in yeast and mammals (7,10,11).…”

“…MGL has demonstrated antitumour efficacy in a number of methionine-dependent cancer cell lines including lung cancer, colon cancer, kidney cancer, brain cancer, prostate cancer, melanoma and fibrosarcoma (7). The antitumor efficacy of MGL, which is based on the greater L-methionine dependency of cancer cells for growth compared to normal cells has been investigated (3). Many human cancer cell lines and primary tumors have an absolute requirement for L-methionine, an essential amino acid (2).…”

“…Thus, depletion of L-methionine is effective for cancer therapy (13). Upon L-methionine depletion, L-methionine-dependent cancer cells are not able to divide and become arrested in the late-S/G2 phase of the cell cycle (2,3,12,14). Cell cycle arrested and death can be reversed by addition of exogenous methionine (12).…”

“…These characteristics have resulted in the development of many enzyme drugs for a wide range of disorders (1). L-Methionine γ-lyase (MGL; EC 4.4.1.11) is a pyridoxal 5'-phosphate (PLP)-dependent multifunctional enzyme which catalyzes α, γ-elimination and γ-replacement of L-methionine and its derivatives and also α, β-elimination and β-replacement of S-substituted L-cysteines (2)(3)(4). This activity was first found using dried cells of E. coli and Proteus vulgaris (4).…”