Biochemistry 1981, 20, 4325-4333
4325
Mechanistic Studies on Reactions of Bacterial Methionine y-Lyase with Olefinic Amino Acidst Michael Johnston,$ Ronald Raines,l Michael Chang," Nobuyoshi Esaki, Kenji Soda, and Christopher Walsh*
ABSTRACT: Methionine y-lyase (EC 4.4.1.1 l), which catalyzes the formation of methanethiol, a-ketobutyrate, and ammonia from L-methionine (eq l), promotes the oxidative deamination of several four- and five-carbon olefinic amino acids (1-5), With the exception of vinylglycine (l), the V , , rates of keto acid formation from the unsaturated substrate analogues are substantially lower than that for processing of methionine to a-ketobutyrate; vinylglycine is deaminated to ketobutyrate and ammonia with a V,,, twice that for L-methionine turnover. and L-2L-Allylglycine, ~-2-amino-3-trans-pentenoate, amino-3-cis-pentenoate (2,4,5) are all converted to 2-ketopentanoic acid (a-ketovalerate). L-2-Amino-3-cis-pentenoate (5) is also a time-dependent, irreversible inactivator of the enzyme. None of the other substrate analogues tested appears to inactivate the enzyme. Spectral analysis of the enzymatic
reaction with cis isomer 5 reveals the formation of a highwavelength chromophore,A( = 550 nm) which implies that a P,y-unsaturated pyridoxal p-quinoid (VI) accumulates. No such absorbing species appears to form during the reaction of trans isomer 4 with methionine y-lyase. But a 550-nm chromophore develops when both 4 and 5 are reacted with Al(NO& and pyridoxal methochloride in methanolic KOH. It would appear that the geometry of the protein and the olefinic amino acid as an intermediate enzyme-substrate adduct controls the kinetics of reaction, such that azaallylic isomerization becomes selectively rate determining for reaction with 5. When this isomerization is slow, an accumulating Michael-type acceptor (VI) could lead to the observed irreversible inactivation of the enzyme.
M i c r o b i a l methionine y-lyase (EC 4.4.1.1 1) catalyzes the formation of a-ketobutyrate, methanethiol, and ammonia from the substrate L-methionine (eq 1). The enzyme has been purified to homogeneity by Soda and his colleagues (Tanaka et al., 1977) and has been shown to be an aZ,& tetramer of four subunits of identical molecular size (mol wt 45 000) but nonidentical in molecular charge (Johnston et al., 1979b). Each subunit contains one residue of tightly bound pyridoxal 5-phosphate (PLP).' The key mechanistic features which account for the oxidative deamination of methionine (eq 1) include pyridoxal-
methionine
ketobutyrate
methanethiol
ammonia
assisted y elimination followed by a net 1,4-azaallylic isomerization (Scheme I). Substrate methionine undergoes (1) the usual transaldimation with enzyme-pyridoxal, (2) a-proton abstraction to yield an a-carbanion equivalent stabilized as a pyridoxaldimine p-quinoid, and (3) 0-proton removal, which leads to the elimination of the y-thiomethyl group and formation of a 0,y-unsaturated p-quinoid (111). Conversion of I11 to IV (the PLP-aminocrotonate derivative) constitutes the azaallylic isomerization step; then reverse t From the Departments of Chemistry and Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 021 39 (M.J., R.R., M.C., and C.W.), and the Institute of Chemical Research, Kyoto University, Uji, Kyoto-Fu 61 1, Japan (K.S. and N.E.). Receiued November 19, 1980; revised manuscript received March 16, 1981. Supported in part by National Institutes of Health Grant GM 2001 1 and by a grant from the American Heart Association. 'National Institutes of Health Postdoctoral Fellow (GM 06430-Ol), 1978-1980. Present address: Department of Chemistry, University of Chicago, Chicago, IL 60637. Massachusetts Institute of Technology Undergraduate Research Opportunities Participant. Present address: Department of Chemistry, Harvard University, Cambridge, MA 02138. II Present address: Merck Sharp & Dohme Research Laboratories, Rahway, N J 07065.
*
0006-296018 110420-4325$01.25/0
transaldimination (step 5 ) forms iminobutyrate and regenerates the cofactor poised for the next catalytic turnover. Hydrolysis to ketobutyrate presumably occurs off the enzyme. Structure I11 is thought to be a key partitioning intermediate which forms during the reaction of all enzymes catalyzing y eliminations and y replacements. It may undergo nucleophilic addition at the C4 vinyl carbon (y replacement), as in the biosynthesis of cystathionine promoted by cystathionine ysynthetase (Davis & Metzler, 1972; Johnston et al., 1979a), or it may isomerize prototropically to the enamino pyridoxaldimine (IV) in the absence of an adding nucleophile, as is suggested for methionine y-lyase above and for y-cystathionase (y elimination; Davis & Metzler, 1972). Direct experimental evidence for the formation of a P,y-unsaturated pyridoxal p-quinoid (111) has been obtained to date only in model organic Abbreviations used: NADH, reduced nicotinamide adenine dinucleotide; KPi, potassium inorganic phosphate; KPPi, potassium inorganic pyrophosphate; LDH, lactate dehydrogenase; PLP, pyridoxal 5phosphate; PNP, pyridoxamine 5-phosphate; TLC, thin-layer chromatography.
0 1981 American Chemical Society
4326
BIOCHEMISTRY
J O H N S T O N ET A L .
reactions (Karube & Matsushima, 1977); in enzymatic systems, its formation as a kinetically competent intermediate has been only inferred. To search for direct evidence for intermediate 111, we have reasoned that each of the olefinic amino acids 1-5 should be substrates for the azaallylic isomerization (111 IV).
-
q C 0 ;
y C 0 ;
"
