3048 served a t -390 nm for the trans CuS2N2 chromophore of
CU(H~NCH~CH~SCH~)~.~CIO~.~~ Acknowledgments. This work was supported by the National Institutes of Health (Grant AM-16412), the Research Corporation, and the Rutgers Computing Center. W e thank Mr. L. Zyontz for assistance with the M U L T A N calculations.
Table 1.O Velocities of Methyl Transfer to DHA from AdoHcyCH3 and AdoHcy-CD3, Catalyzed by COMT 1 0 9 1 / ( ~min-’)*
1OS[AdoMet], M 3.86 7.73
References and Notes J. A. Thich, D. Mastropaolo, J. Potenza, and H. J. Schugar, J. Am. Chem. Soc., 9S, 726 (1974). J. R. Wright and E. Frieden, Bioinorg. Chem., 4, 163 (1975), and references therein. W. M. Weigert, H. Offermanns, and P. Scherberich, Angew. Chem.. Int. Ed. Engl., 14, 330 (1975). H. C. Freeman and P. J. Birker, personal communication. F. I. Carroll, J. D. White, and M. E. Wall, J. Org. Ch8m., 28, 1240 (1963). Anal. Calcd for Cu,4[SC(CH3)2CH2NH2] 12CI.3.5S04.19H20: Cu, 31.17; S, 17.41; N. 5.89; C, 20.20; H, 5.58; CI, 1.24; S (as SO,). 3.93; Zn, 0.0. Found: Cu, 31.35, 31.27, 31.88; S,17.99, 17.80; N. 5.84. 6.05; C, 20.31, 21.08; H, 5.40, 5.31; CI, 1.26, 1.28; S (as SO‘), 4.31; Zn, 0 to “trace”. The crystals grow as twins, and apparently dehydrate if not sealed in a capillary with some DMFIH20 mother liquor. Program MULTAN, P. Main, M. M. Woolfson, and G. Germain, Department of Physics, University of York, England. The discrepancy between dobd and dcalcd has not been resolved by our attemDts to minimize Sample dehydration;’ the presence of additional H&, DMF,’or other lattice species is not indicated by elemental anal ses R F = ZIIFd - I F d l I x l F d : R w F = [ E d F d - I F d ) 2 / X ~ ~ d y 1 1 / 2 : This formulation agrees with the results reported‘ for c%nplex II and is supported by the special chemical role of CI- in the formation of 11.2*12 An alternate formulation with S2- as the central ion and CI- as a disordered lattice species cannot be ruled out entirely. W. K. Musker and C. H. Neagley, Inorg. Chem.. 14, 1728 (1975). G. Nickless. Ed., “lnorqanic Sulfur Chemistry”. Elsevier, New York, N.Y.. 1968, pp 724-726. F. J. Hollander and D. Coucouvanis, J. Am. Chem. SOC.,96, 5646 (1974), and references therein. M. B. Robin and P. Day, Adv. Inorg. Radiocbem., I O , 247 (1967). C. Sigwart, P. Hemmerich. and J. T. Spence. Inorg. Chem., 7,2545 (1968). Similar spectra were reported by E. W. Wilson, Jr., and R. B. Martin, Arch. Biochim. Biophys., 142,445 (1971). D.R. McMillin, R. C. Rosenberg. and H. B. Gray, Roc. Natl. Acad. Sci. U.S.A., 71, 4760 (1974). Our structural and spectroscopic studieslg of approximately T, C O [ S C ( C H ~ ) ~ C H ~include N H ~ ] ~the identification of the S Co(ll) 6- and a-LMCT bands. These results along with our cluster studies support the assignments by McMillin et al. of the Cu(ll) and Co(ll) protein spectra. D. Mastropaolo, J. A. Thich, B. Cohen, J. Potenza. and H. J. Schugar, to be submitted for publication. C. Ou, B. Vasiliou, V. Miskowski, J. A. Thich, R. A. Lalancette. J. A. Potenza, and H. J. Schugar, to be submitted for publication.
‘
11.59 15.45 38.63 103.0
A~oHcv-CH~ 1335 7,977 i 11 1922 f 7, 1854 f 7, 1823 f 6 2011 f 5,2024 f 5 2282 f 5,2307 f 5, 2280 f 4 2436 f 6,2545 f 6 2728 f 11.2361 f 8
AdoHcv-CD? 2049 f 6,2021 f 6, 1957 f 8 2386 f 5,2290 f 5 2659 f 4,2597 f 5 , 2597 f 5 2813 f 7,2838 f 9 3024 f 12. 3155 f 11
Rates measured at 360 nm, 37.00 f 0.05’, phosphate buffer (0.125 M), pH 7.6, [Mg*+] = 1.5 X M, [DHA] = 2.5 X M, [dithiothreitol] = 4.5 X M, protein = 0.839 mg/ml. Velocities in M min-’ were calculated from d(absorbance)/dt using A360eff = 2877. Error limits are standard deviations within a single run. AdoMet
+
DHA
COMT
m2+
S-adencqlhomocysteine ( AdoHcy)
OCHB
+ @OH+++ COCHj
(1)
COCH,
-
1, AdoMet or AdoHcy-CL,
COCH,, 2, DHA
H. J. Schugar,* C. Ou, J. A. Thich, J. A. Potenza* School of Chemistry, Rutgers University New Brunswick, New Jersey 08903 R. A. Lalancette, W. Furey, Jr. Department of Chemistry, Rutgers University Newark, New Jersey 07102 Received February 20, 1976
SNZ-Like Transition State for Methyl Transfer Catalyzed by Catechol- O-methyltransferasel Sir: The velocity of methyl transfer (eq 1) from S-adenosylmethionine (AdoMet or AdoHcy-CL3, 1, L = H or D) to 3,4-dihydroxyacetophenone (DHA, 2), catalyzed by ratliver catechol-0-methyltransferase (COMT),* is increased substantially by trideuteration a t the transferred methyl group (VH/VD = 0.832 f 0.045 a t 37.00 f 0 . 0 5 O ) . This inverse a-deuterium secondary isotope effect is exactly what is observed for many classical s N 2 reactions in organic-reaction system^,^ and constitutes a strong indication that C O M T catalysis involves rate-limiting s N 2 methyl transfer with a trigonal-bipyramidal transition state of the type shown in structure 3. This information should prove critical
J o u r n a l of the American Chemical Society
/
98:lO
3
for efforts now in progress4 to design transition-state-analogue inhibitors, in part for use as drugs, of this important enzyme and closely related enzymes of the liver and central nervous ~ y s t e m . ~ Table I shows velocities of methyl transfer by C O M T a t various concentrations of AdoHcy-CH3 and AdoHcy-CD3. T h e data for the CH3 cofactor generate the MichaelisMenten expression of eq 2, while that of eq 3 is produced by the rates for the CD3 cofactor. 1 0 9 1 / ~( M min-I) = (2760 f 90)[AdoMet]/([AdoMet] (4.1 f 0.6) X (2)
+
1 0 9 V (~M min-’) = (3220 f 60)[AdoMet]/([AdoMet] (4.3 f 0.4) x 10-51 (3)
+
Absorbance changes at [AdoMet]
