3975
T. C. FARRAR, H. S.GETOWSKY, R.A. ALBERTYAND W. G. XILLER
[ CONTRIBUTIOX FROM
THE S O Y E S
CHEMICAL LABORATORY, USIVERSITY O F ILLINOIS, UNIVERSITY O F WISCONSIN]
A S D THE
Vol. 79
DEPARTMENT O F CHEMISTRY,
The Mechanism of the Stereospecific Enzymatic Hydration of Fumarate to L - M a l a t e 1 BY T. C. FARRAR, H. s. GtiTO\%rSIiY, R. A. ALBERTY A I ~ DW. G. XILLER RECEIVEDFEBRUARY 7, 1957
It is found t h a t the addition of water to the double bond of fumarate occurs in a cis manner when catalyzed by the enzyme fumarase from pig heart muscle. This is shown by the configuration of the monodeutero-L-malate formed when the hydration is carried out with deuterium oxide. The configuration of the monodeutero-L-malate was established by observing the dipolar broadening of the proton magnetic resonance absorption in solid DOOCCH(D)C(OD)HCOOD.
Introduction other sources would necessarily cause deuterium 'CVhen the hydration of fumarate to L-malate is to be added in the same position as pig heart fucatalyzed in deuterium oxide by the fumarase from marase. The question of whether the fumarase catalyzed pig heart muscle, the L-malate which is formed hydration occurs in a cis or trans manner can be contains one deuterium atom per molecule which answered by establishing the steric relation of the is not readily exchangeable with the Since only monodeutero-L-malate is formed in the fumarate protons on carbons 2 and 3 in the monoover-all reaction and the fumarate does not incor- deutero-L-malic acid. This is shown schematically porate deuterium, the addition of the deuterium in the right-hand portion of Fig. 1. If the hydrais stereospecific, as is the addition of the OH or OD tion occurs in the cis manner as depicted in the group. This observation has been ~ o n f i r m e d . ~figure, the protons are gauche to one another. On Further work has shown t h a t the deuterium atom the other hand, if the hydration is trans, then the is not exchanged with the medium by the action of protons are trans. These particular relations the enzyme but is removed only in the over-all de- hold only if the carboxyl groups are trans in L-malic acid, as shown; if the carboxyl groups are gauche, hydration reaction. The question as to which of the two positions on then the proton-proton relations are the reverse of the methylene group is deuterated in the mono- those just given. X-Ray diffraction studies of succinic and tartaric deutero-L-malate is important because the answer acids8 have shown that in the crystalline solids the will show whether water is added to the double bond of fumarate in a cis or trans manner. \{There- carboxyl groups are trans, and undoubtedly they as it frequently is assumed that substrates are are also trans in crystalline malic acid. Therefore, bound on the surface of an enzyme, a cis addition the only structural feature to be established is by fumarase would prove t h a t the entering groups whether the protons are gauche or trans in the came from the same side, and presumably from monodeutero-L-malic acid. This type of structural that of the protein since their addition is under problem is suitable for proton magnetic resonance 5tereochemical control. The ability of the enzyme solid-state methods. The hydroxyl and acidic to distinguish unerringly between the two hydrogen protons exchange readily with D 2 0 to give DOpositions on the methylene group of L-malate may OCH(D)C(OD)HCOOD, in which the protons in find practical application in the determination of question are the most important magnetic nuclei. the structure of partially deuterated compounds The dipolar broadening of the proton magnetic resonance in the solid depends very largely on the which may be transformed to malic acid. The stereospecificity of the diphosphopyridine intramolecular proton-proton distance. The broaddehydrogenases already has been i n ~ e s t i g a t e d . ~ening is very sensitive to the interproton distance I t has been found that the hydrogen transfer cat- so it is relatively easy to determine unequivocally alyzcd by the 0-hydroxysteroid dehydrogenase6 whether the configuration is fraizs or gauche, as has and glucoze dehydrogenase' occurs a t the opposite been done for solid sym-tetrachloro- and tetrabroside of the nicotamide ring from that used by the r n ~ e t h a n e . ~ alcohol, lactic and malic dehydrogenases. ThereExperimental fore, it is not to be assumed that fumarase from Preparation of Deutero-1,-malic Acid.-Crystalline fu(1) T h c nuvlear magnetic resonance expesiments u-ere supyrxted b y the U. S Oilice of Naval Research and b y a grant-in-aid from the (Tjijohii C < , , T h e enzymatic experiments were supported by grants from t h e S a t i o n a l Science Foundation and from t h e Research Committee of t h e Graduate School of t h e University of Wisconsin from f u n d i snpplied by t h e Wisconsin Alumni Research Foundation, ( 2 ) €1, Fischer. C. Fprieden, J. S. McKinley hicKee and R. A. Alberty, THISJ O U R N A L , 7'7, 4436 (1935). i.3) S. Englard and S Colowick, J . Biol. Chem.. 221, 1019 (1956). (4) R . A. Alberty, XV. G. hiiller and H. Fisher, T H I s J O U R N A L , 79. 3973 (1937) (ri) B. Venriesland and F. Westheimer, in \V. D . UcElroy and B. Glass, "The 3lechanism of Enzyme Action," Johns Hopkins Press, Baltimore, 3 I d , 19.54. (I;) P. Talalay, F. A . Loewus and R . Vennesland, .I. Biol. Che77i.. 212, 801 (1933). (7) FI. R. Levy, F. A . 1,oewus and R . Vennesland, ibid.,222, G8.j (I!J.if,)
marase obtained from pig heart estract1O was added to a solution containing 0.2 251 potassium fumarate and 0.0083 JI dipotassium phosphate and 0.0017 -11monopotassium phosphate in 99.5y0 D20i1 and the reaction was allowed to proceed t o equilibrium, as determined spectrophotometrically. The reaction was stopped by heating t o 90" for 3 minutes and the solution was then lyopliilized. T h e deutero-L-malate was separated from the 0.18 mole fraction fumarate present a t equilibrium bv partition chro(8) R. W. G. Wyckoff, "Crystal Structures," Interscience Publishers, Inc., New York, S . I-., 1953. Vol. 11, Chap. I I I I C . (9) M. Takeda and H. S . Gutowsky, 3. Chem. P h y s . , 26, 577 (1957). (IO) C. Frieden, R. 11. Bock a n d I
