ABSTRACT
In the spatial structure of methionine γ-lyase (MGL, EC 4.4.1.11) from Citrobacter freundii, Tyr58 is located at H-bonding distance to the oxygen atom of the phosphate "handle" of pyridoxal 5'-phosphate (PLP). It was replaced for phenylalanine by site-directed mutagenesis. The X-ray structure of the mutant enzyme was determined at 1.96Å resolution. Comparison of spatial structures and absorption spectra of wild-type and mutant holoenzymes demonstrated that the replacement did not result in essential changes of the conformation of the active site Tyr58Phe MGL. The Kd value of PLP for Tyr58Phe MGL proved to be comparable to the Kd value for the wild-type enzyme. The replacement led to a decrease of catalytic efficiencies in both γ- and ß-elimination reactions of about two orders of magnitude as compared to those for the wild-type enzyme. The rates of exchange of C-α- and C-ß- protons of inhibitors in D2O catalyzed by the mutant form are comparable with those for the wild-type enzyme. Spectral data on the complexes of the mutant form with the substrates and inhibitors showed that the replacement led to a change of rate the limiting step of the physiological reaction. The results allowed us to conclude that Tyr58 is involved in an optimal positioning of the active site Lys210 at some stages of γ- and ß-elimination reactions. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.
Subject(s)
Carbon-Sulfur Lyases/chemistry , Citrobacter freundii/enzymology , Carbon-Sulfur Lyases/metabolism , Catalytic Domain , Kinetics , Magnetic Resonance Spectroscopy , TyrosineABSTRACT
Methionine γ-lyase (MGL) catalyzes the γ-elimination of l-methionine and its derivatives as well as the ß-elimination of l-cysteine and its analogs. These reactions yield α-keto acids and thiols. The mechanism of chemical conversion of amino acids includes numerous reaction intermediates. The detailed analysis of MGL interaction with glycine, l-alanine, l-norvaline, and l-cycloserine was performed by pre-steady-state stopped-flow kinetics. The structure of side chains of the amino acids is important both for their binding with enzyme and for the stability of the external aldimine and ketimine intermediates. X-ray structure of the MGL·l-cycloserine complex has been solved at 1.6 Å resolution. The structure models the ketimine intermediate of physiological reaction. The results elucidate the mechanisms of the intermediate interconversion at the stages of external aldimine and ketimine formation.
Subject(s)
Bacterial Proteins/chemistry , Carbon-Sulfur Lyases/chemistry , Citrobacter freundii/chemistry , Imines/chemistry , Pyridoxal Phosphate/chemistry , Alanine/chemistry , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/genetics , Carbon-Sulfur Lyases/antagonists & inhibitors , Carbon-Sulfur Lyases/genetics , Catalytic Domain , Citrobacter freundii/enzymology , Crystallography, X-Ray , Cycloserine/chemistry , Cysteine/chemistry , Enzyme Inhibitors/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Glycine/chemistry , Kinetics , Models, Chemical , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Thermodynamics , Valine/analogs & derivatives , Valine/chemistryABSTRACT
Pyridoxal 5'-phosphate (PLP) dependent methionine γ-lyase catalyzes the breakdown of L-methionine to α-ketobutyric acid, methanethiol and ammonia. This enzyme, present in anaerobic microorganisms, has biomedical interest both for its activity as antitumor agent, depleting methionine supply in methionine-dependent cancers, and as target in the treatment of human pathogen infections, activating the pro-drug trifluoromethionine. To validate the structure of the enzyme from Citrobacter freundii, crystallized from monomethyl ether polyethylene glycol 2000, for the development of lead compounds, the reactivity of the crystalline enzyme towards L-methionine, substrate analogs and inhibitors was determined by polarized absorption microspectrophotometry. Spectral data were also collected for enzyme crystals, grown in monomethyl ether polyethylene glycol 2000 in the presence of ammonium sulfate. The three-dimensional structure of these enzyme crystals, solved at 1.65Å resolution with R(free) 23.2%, revealed the surprising absence of the aldimine bond between the active site Lys210 and PLP. Different hypothesis are proposed and discussed in the light of spectral and structural data, pointing out to the relevance of the complementarity between X-ray crystallography and single crystal spectroscopy for the understanding of biological mechanisms at molecular level. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.
