May 20, 1957
COMMUNICATIONS TO THE EDITOR
bumin. When the sulfhydryl group in denaturing medium is inactivated by addition of 0.68 mole of silver nitrate or potassium ferricyanide, the reaction ratio of copper(I1) .to BSA was again found to be unity. Our experiments have been carried out under conditions quite different from those of Klotz, et a1.,4 and no comparison with their results is made here. Our results show t h a t in the pH range 8-10, one copper(I1) is bound to the BSA molecule extremely tightly, and t h a t the sulfhydryl group is not the reactive group. It is of interest to note that nickel(II), even in very small concentrations, and also, to a lesser extent, cobalt(I1) greatly interfere with the copper(I1) BSA reaction while even large amounts of zinc(I1) have very little effect. Thus, the group in the BSA molecule responsible for the binding of copper(II), the nature of which is a t present unknown, apparently also forms stable complexes with nickel(I1) and cobalt(I1) but not with zinc(I1). One unique site in the BSA molecule is the N-terminal aspartyl group, and further work may show whether this group is the site of copper(I1) binding. There is very little (if any) reaction between copper(I1) and human 7-globulin under our conditions. The method has been applied to the determination of albumin in blood serum, and preliminary results are promising. Acknowledgment.-This work was supported by grants from the U. S. Public Health Service and from the Louis and Maud Hill Family Foundation. (4) I. M. Klotz and H. G. Curme, THISJOURNAL, 70, 939 (1948); H.A. Fiess and I. M . Klotz, ibid., 74, 887 (1952); I. M. Klotz. J. M. Urquhart and H. A. Fiess, ibid., 74, 5537 (1952); I. M .Klotz, J. M Urquhart, T. A. Klotz and J. Ayers, ibid., 77, 1919 (1955); I. M. Klotz, I. L. Faller and J. M. Urquhart, J . Phys. Colloid Chem., 64, 18 (1950); I. M. Klotz and H. A. Fiess, ibid., 66, 101 (1951).
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The sequence of a portion of the active site of chymotrypsin has been e ~ t a b l i s h e d ~and - ~ hence a comparison could be made if similar information could be obtained for an enzyme of very different specificity. Such an opportunity arose when it was demonstrated that the active site of phosphoglucomutase was marked by a serine pho~phate.’,~ P32-labeled enzyme was prepared by exchange with radioactive substrates ( g l ~ c o s e - l - P O 4and ~~ g l ~ c o s e - 6 - P O ~under ~ ~ ) conditions similar to those of the standard assay.g I t had been established that the enzyme is labeled only a t the active site by this procedure.8 The enzyme was then degraded, either with acid alone, with proteolytic enzymes alone, or with proteolytic enzymes followed by acid hydrolysis. Radioactive phosphopeptides were isolated by use of Dowex 50 columnsl’J.ll and paper chromatography. The compositions of the phosphopeptides are shown in Table I. P
a
2
TABLE I P ISOLATED ~ ~ FROM ~
-
PHOSPHOGLUCO~ ~ ~ ~ ~
MUTASE
Peptide
Phosphopeptide obtained by treatment with
+ + +
5
Proteolytic acid Proteolytic acid Proteolytic acid Proteolytic only Proteolytic only
6 7
Acid only Acid only
1
2
3 4
Amino acid compn.
(Asp,Ser,Gly,Glu) (Asp,Ser,Gly,Glu,Ala)
(Asp,Ser,Gly,Glu,Ala,Val,Thr) (Asp,Ser,Gly,Glu) (Asp,Ser,Gly,Glu Ala,Val,Thr, Leu) (Asp,Ser,Gly,Glu,Ala) (Asp,Ser,Gly,Glu,Ala,Val Thr, Leu)
While the detailed sequence has not been completely established, it is to be noted that the com(5) On leave of absence from Bucknell University, Lewisburg, Pa. position of each peptide and the order from comSCHOOL OF CHEMISTRY parison of lower and higher peptides is in perfect UXIVERSITY OF MINNESOTA I. hl. KOLTHOFF h!fINNEAPOLIS 14,MISN. B. R. WILLEFORD,J R . ~ agreement with the sequence for chymotrypsin which is Asp Ser Gly Glu Ala Val.4-6 The probRECEIVED MARCH25, 1957 ability t h a t the 7 peptides isolated from phosphoglucomutase would agree with this sequence ENZYME CATALYSIS AND ENZYME SPECIFICITYby chance alone is very small. Moreover the conCOMBINATION O F AMINO ACIDS AT T H E ACTIVE cordance of the acid derived and proteolytic enSITE OF PHOSPHOGLUCOMUTASE1 zyme plus acid derived peptides is strong evidence Sir : against the sequence being an artifact of the sepaIn the classical template model for enzyme action ration procedure. The data therefore indicate that the substrate is assumed to be absorbed with a pre- the amino acid sequence of the active site for a t cise fit into an area called the “active site” of the least 6 amino acids is the same for an enzyme which protein.2 Presumably the amino acids a t this site specifically hydrolyzes peptide bonds and an enprovide not only the catalytic action but also the zyme t h a t specifically transfers phosphate bespecificity of the enzyme. From the widely varying tween carbohydrate molecules. specificity patterns of enzymes it would be anticiThis apparently surprising result is good suppated t h a t the amino acid composition a t the port for the above mentioned hypothesis that comactive site would vary to fit the particular sub- mon bond-breaking mechanisms exist despite varystrate. On the other hand, stereochemical and (4) J. A. Cohen, R. A. Oosterbann, M. G. Warringa and H. S. other evidence had led to the hypothesis that a few Jansz, Disc. F a r a d a y Soc., 20, 114 (1955). bond-breaking mechanisms might be common to (5) N. K.Schaffer, S. Harshman, R. R. Engle and R. W. Drisko, reactions of widely different specificities3 and hence Federation Proc., 14, 275 (1955). (6) F. Turba and G. Gundlach, Bid. Z., 327, 186 (1965). there might be some common amino acid sequences. (7) G.R. Jolles and L. Anderson, Am. Chem. SOC.Abstracts, Sept.
(1) Research carried out at Brookhaven National Laboratory under t h e auspices of t h e U. S. Atomic Energy Commission. ( 2 ) E. Fischer, Ber., 27, 2985 (1894). (3) D. E. Koshland, Jr., “Mechanism of Enzyme Action,” edited by McElroy and Glass, Johns Hopkins Press, Baltimore, Md., 1954, p. 608.
1955, p. 232. (8) E. P. Kennedy and D . E. Koshland, Jr., J. Bid. Chem., in press. (9) V. A. Wajjar, ibid., 176, 280 (1948). (10) M.Flavin, ibid., 210, 771 (1954). (11) N. K. Schaffer, S. Harshman and R. R. Engle, ibid., 214, 799 (1955).
~
~
2658
COMMUNICATIONS TO THE EDITOR
Vol. 7 ! )
ing specificity type. I t suggests further that (a) the common sequence around serine is involved in making the otherwise inert CH20H side chain reactive and (b) the amino acids responsible for the specificity lie further from the reactive serine, perhaps in adjacent coils. \Vhile there must be an intimate steric relation between the amino acids responsible for the catalytic action and those rcsponsible for the specificity, these results indicate that the two may be separable. In this case conimon sequences related to specificity as well as to the bond-breaking action might be expected, a result which would be of enorinoiis value in relating enzyme structure to function.
acetone and the incubation continued for 24 hours longer. Extraction of the culture with ethyl acetate, followed by chromatography on silicic acitl . crystallization from hexane-acetone, and sub1ini:itioii a t 90b11.5° (0.000,5 mrn.) afforded 20.3 xng. o f IT', n i . ~ ) 140.,j-141° . (no depression on adniixturr. with an authentic sample); [ a ] ? b+ ~1 2 3 O (c 1.04. in CHC1:J; XE:P, 243 mp ( E 16,200); sulfuric ncj(1 chromogenss were identical with a known sariiplc (peaks a t 2G5, 305 and 390 (broad) and a minilnuin a t 2% nip); (Anal. Found: C, R0.08; H, S.16. Calcd. for CI9Hz4O2:C , 80.21; H, S.51); infrared analysis showed Agfl 5.77 p (17-ketone), 6.04, 6.17 arid 6.25 ,u (A1*4-3-ketone),identical with an ailtheritic sample. Mixed paper chromatography BIOLOGYDEPARTMENT BROOKHAVEN YATIOSAL L.4BORAToRY D. E. KOSHLAND, JR. showed no separation from authentic 1x7. UPTON,L. I . , KEWYORK MARYJANE E R U I Y In similar experiments, conversions in good yield RECEIVED APRIL 15, 1957 of I and 111 to IV were demonstrated and the products rigorously identified by comparison paper ENZYMATIC INTRODUCTION OF DOUBLE BONDS chromatography, m.p. and infrared spectra. Evidence for the presence of small amounts of V in each INTO STEROID RING A case was obtained. Conversion of 1S-nor-testosSir: terone by the same system in 72 hours gave a 67 ' The introduction of unsaturation into ring A of yield of estrone 1 n . p . 253--236'; [a]"D f 1.isteroids is of interest in connection with the aro- (c 0.644, in dioxane); 28%m,u ( E 1960); on admatization involved in biosynthesis of estrogens, dition of XaOH, 243 m p ( E 8000) and 300 nip and in the microbiological formation of A'-dehy- ( 6 2500). The infrared spectrum showed peaks a t drosteroids, some of which possess interesting
