BOOKREVIEWS
May 20, 19%
2915
CO in the ratio 2 C U + / C O ,hydroxylation ~ by this TABLE I COMPARISON O F OL8 I N 4,5-DIMETHYLCA4rECHOLFORMEDenzyme system is describable as ENZYMICALLY F R O M 3,4-DIMETHYLPHEYOLa I N 018? AND cu+ 02 +P r o t e i n (cu-.,_ cu*zr~ Hz0 AND I N 0 2 A N D H,OLsC (1) Protein(Cu
+
Experiment
O'%
+ HzO
Found
Atom 9% excess O l s d theoretical for uptake of one a t o m
No uptake
0.52 0.59 0.00 .51 .56 o2 rlzo18 .00 0.59 0.00 . 00 Twenty-five ml. reaction volumes contained 0.3 minolc ascorbic acid, 1.3 mmole KHzPOa, 2.15 mmole KzHPOI,0.45 mrnole 3,4-dimethyIphenol and 4.0 mg. purified5 mushroom phenolase having 20-50 cresolasea and ca. 1000 catecholase' units/mg. dry wt. 4,5-Dimethylcatechol (3C-50% yield) was isolated through its lead salt, from an ether extract of the reaction mixture, m.p. 84-86'. No hydroxylation occurred in the system when heat-denatured enzyme was substituted for active protein. * Prepared electrolytically. Obtained from the Stuart Oxygen Company, containing Mass spectrometry was performed by 1.4 atom % O'*. the Consolidated Engineering Corporation on carbon dioxide samples obtained by Unterzaucher pyrolysis* of 4,5-dimethylcatechol samples. Oxygen recovery was quantitative.
cu-- ..
(2) Protcin(C
0-0
p-*-
+
Since the phenolase complex is a cuprous proteinY~10,11~12 which is in the cupric form after each h y d r ~ x y l a t i o nand l ~ which combines with inhibitor (5) RI. F. Mallette and C. R. Dawson, Arch. Biochem., 23, 29 (1919). (6) M . F. hlallette and C. R . Dawson, THISJOURNAL, 6 4 , 2344 (1942). ( 7 ) W. H. Miller, hl. P. Mallette, L. J. R o t h and C. R . Dawson, ibid., 66, 514 (1944). (8) W. E. Doering and E. Dorfman, ibid.,7S, 5595 (1953). (9) P. Kuhowitz, Biocheni. Z., 292, 221 (1937); 299, 32 (19)38), c f . D. Keiiin, Proc. Roy. Soc., ( L o d o n ) , 104B, 206 (1929); D Keilin and T. M a n n , ibid., l2SB, 187 (1938). (10) J . Doskocil, Coliccfion Cecckosloo. Chcin. Coinrnun., 16, 61 4 (1950). (11) A. B. Lerner, "Advances in Enzy~nology,"Val. X I V , F. P'. Nord. ed., 1968, p. 73. (12) H . S. Mason. "Advances in Enzymology." VoI. X V I , F. I?. Nord, ed., 1955, p. 105. (13) Hydroxylation does not proceed in t h e absence of reducing agents: c f . R. C. Behm and J. M. Nelson, THISJ O U R N A L , 66, 711 (1944); M. Suda, N. Kimoto a n d S. Naono, J . Biochciir. SOL.( J a p a n ) , 26, 603 (1954); A. B. Lerner, T. B. Fitzpatrick, E . Calkins and W. H.
+ Monophenol
Protein(Cu"
--f
+ Diphenol + Hz0
cu++
cu-r+ (3) Protein(Cu
++
+ 2e-
---+
:I
