in addition to undergoing reactions (3b), C6HbS. iuoiiodeutero-L-malate which is produced enzycnters into two termination processes : matically made it possible to determine whether the breaking of the methylene carbon-hydrogen bond CGHLS. (C&&CH. --f ( C G H ~ ) ~ C H S C(V) ~ H S(4a.j is the rate determining step in the enzymatic 2 CGH~S. --+ CsHsSSCsHs (VI) (4b) The infrared spectrum of residues after isolation reaction. The breaking of the methylene carbondeuterium bond in deutero-1,-malate must proceed of I11 was identical with that of a solution prepared a t approximately one-sixth the rate of that for the and VI.7 Evi- corresponding from an authentic sample of carbon-hydrogen bond.5 If the dence for the occurrence of reaction (3b) was found breaking of a carbon-hydrogen bond were rate in the formation of tetraphenylethane 111 in ‘727; limiting, the over-all rate of reaction would be yield by exposure of a dilute solution of diphenyl decreased by a factor of six in the dehydration of disulfide VI in diphenylmethane to a sun lamp. deutero-L-malate. Studies with S35 have shown that disulfides disWhen monodeutero-L-malate was dehydrated sociate into mercaptyl radicals on irradiation.8 enzymatically a t pH 8.0 in 0.005 M tris-(hydroxylilank experiments indicated that compounds IV, methyl)-aminomethane perchlorate buffer it was 1. and VI did not cause formation of I11 from 11, found that both the maximum initial velocity and nor did they adversely affect the isolation of I11 Michaelis constant were unchanged after a correcfrom II.9 tion for a small amount of fumarate in the malate ( 6 ) C. Fingi and V. Bellavita, G a m chim. i f a l . , 62, 699 (193?). preparation. Thus the breaking of the carbon-(7) T. Zinke and W. Frohneberg, Bsv., 43, 840 (1910). hydrogen bond is not involved in the rate-deter( 8 ) E. N. Guryanova and V. N. Vasileva, Zhui.. F i z . Khinz., 23, GO mining step. It seems likely from kinetic argu(1954). (9) This work was supported by a grant from the S a t i o n a l Science m e n t ~ ~that . ’ the dissociation of furnarate from the Foundation. enzyme is not the rate-determining step for the SCHOOL OF SCIENCE dehydration reaction at high L-malate concentraBRANDEIS UNIVERSITY SAUI, G. C O H E N tions. C1rr F1r.A M ’ A N G \\‘AI,THAM 64, hf ASSACMUSETTS The fact that the deuterium of monodeutero-1.RECEIVED JUSE 11, 1953 malate was removed in a step which is not rate limiting suggested t h a t there should be a sterically CONCERNING THE STEREOSPECIFICITY OF THE specific exchange of this hydrogen atom proceeding FUMARASE REACTION AND THE DEMONSTRATION at a rate faster than the dehydration reaction. OF A NEW INTERMEDIATE’ Such a rapid exchange was indeed found to occur Sir: in an experiment in which L-malate was dehydrated When fumarase catalyzes the addition of water enzymatically in 99.5% D20to the extent of 0.04%. to fumarate only the L-isomer of malate is pro- The amount of deutero-L-malate which could duced.2 While the hydroxyl group is added have been formed by the reverse reaction from the stereospecifically, the question still arises as t o the fumarate so produced and initially present in the stereospecificity of the addition of the hydrogen sample would be immeasurably small. However, atom. Vb7e have been able t o demonstrate that the dipotassium malate recovered had incorporated this process is absolutely stereospecific. Di- about 0.003 atom of deuterium per molecule. potassium fumarate was added to a reaction mix- This relatively rapid exchange demonstrates the ture containing crystalline fumarase, 0.038 M existence of an intermediate in which the hydrogen K2HP04, and 0.015 I, I.eigh, T H I J~ OUHNAI.. 1 4 , 5933 (195?).
