| Popis: |
We have studied and compared the pH-dependencies of the main kinetic parameters for the α,γ-elimination reactions of methionine γ-lyase (MGL) of Citrobacter intermedius with natural substrate, l -methionine, with its phosphinic analogue, and for α,β-elimination reaction with S-methyl- l -cysteine. From the pH-dependency of k cat / K m for the reaction with l -methionine we have concluded that MGL is selective with respect to the zwitterionic form of its natural substrate. For the reaction of MGL with 1-amino-3-methylthiopropylphosphinic acid the p K a of the substrate's amino group, equal to 7.55, is not reflected in the pH-profile of k cat / K m . Consequently, the enzyme does not manifest well-defined selectivity with respect to the zwitterion and anion ionic forms of the substrate. The ascending limbs of pH-dependencies of k cat / K m for reactions with l -methionine and S-methyl- l -cysteine are controlled by a single p K a equal to 7.1–7.2, while for the reaction with 1-amino-3-methylthiopropylphosphinic acid two equal p K a s of 6.2 were found in the respective pH-profile. The descending limbs of pH-dependencies of k cat / K m for the reactions with S-methyl- l -cysteine and racemic 1-amino-3-methylthiopropylphosphinic acid are very similar and are controlled by two acidic groups having average p K a values of 8.7. On the basis of these results we suggest a mechanism of catalytic action of MGL. According to this mechanism Tyr 113, in its conjugated base form, acts as an acceptor of the proton from the amino group of the substrate upon its binding in the active site. Elimination of the leaving thiol groups during both α,γ- and α,β-elimination reactions is assisted by the acidic groups of Tyr 113 and Tyr 58. Both tyrosyl residues are able to fulfill this catalytic function with different efficiencies depending on the type of elimination reaction. Tyr 113 residue plays the determining role in the α,γ-elimination, and Tyr 58 — in the α,β-elimination process. |
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