Sept. 5, 1960
COMMUNICATIONS TO THE EDITOR
heated a t 120-130”, the ethylenimine VI11 was formed in 5570 crude yield (from IV), m.p. 153l54”, or isolated as a second crystal form, m.p. 143-145’ (Found: C, 63.4; H, 6.60; N, 5.20); N H absorption a t 3.05 p. By treatment with carbon disulfide and iodomethane under the conditions used to prepare 11, compound VI11 was converted back to IV, and with benzoyl chloride it gave the N-benzoyl derivative, m.p. 195-198” (Found: C, 68.1; H, 5.50; N, 3.95). When the anhydromannoside Vl0 was heated with excess ammonium thiocyanate in 2-methoxyethanol a t 105-1 lo”, the crystalline thiocyanohydrin VI was formed in 75-90Oj, crude yield, m.p. 188-190” (Found: C, 55.8; H , 5.30; S, 9.61). Opening of the epoxide ring of V a t C.3 to give the trans-diaxial product VI as the predominant product is assumed by analogy with other nucleophilic cleavages of I. The conformational rigidity imposed by the 4,6-benzylidene ring obviously prevents the direct epoxide-episulfide transformation that commonly results from the action of thiocyanate ion on epoxides.ll Mesylation of VI gave the sulfonate ester VI1 as a foam in 92% crude yield and treatment of VII, dissolved in 2methoxyethanol, with aqueous sodium hydroxide12 resulted in a 62% yield of the episulfide IX, m.p. 166” (Found: C, 60.2; H, 5.79; S, 11.5). The potentially wide utility of VI11 and I X for further synthetic transformations to compounds such as aminomercapto sugars and diamino sugars which may have interesting chemotherapeutic properties is under investigation. The authors wish to thank Dr. B. R. Baker for valuable suggestions.
4739
100 90
80 70
_’’ A L L- 0 R - NONE ” ASSAY
60
k = O 38 M I N - ’ 50 G-I-P+E-P 0
5
0
40
32
G - 6 - P + E-P”
a z W LL
-RATE
> 30 k
ASSAY
k : 0 96 MIN-’
z
t-
u
a
s 20
\ \ \ \
\
IO
0
I 2 3 PHOTOOXIDATION TIME IN M I N U T E S
4
Fig. 1.-Loss of phosphoglucomutase activity on photooxidation as measured by the “rate” assay (A) and the “all-or-none” assay ( 0 ,0 ) .
jiciency of all enzyme species present. The other assay, an “all-or-none” assay, was different in (1945). that i t measured that fraction of the enzyme capable (11) E. E. van Tamelen, THISJOURNAL, 73,3444 (1951). of functioning a t all. To do this, P32-labeled (12) L . Goodman a n d B. R. Baker, i b i d . . 81, 4924 (1969). enzyme was incubated with a large excess of G-1-P DEPARTMEKT OF BIOLOGICAL SCIENCES (or G-6-P) for time intervals sufficient to allow STASFORD RESEARCH INSTITUTE JLMES E. CHRISTESSEN ample opportunity for the intact enzyme to unMESLO PARK, CALIFORNIA LEONGOODMAN dergo a t least 730 complete turnovers. A modified RECEIVED AUGUST4, 1960 enzyme retaining as little as 1/200 of its original activity would therefore lose essentially all its P32-labelduring the assay. ROLE OF HISTIDINE IN PHOSPHOGLUCOMUTASE. The activity remaining as a function of photoT H E USE OF “RATE” AND “ALL-OR-NONE” ASSAYS oxidation time measured by these two assays is sir: shown in Fig. 1. The first order rate constants for The use of chemical modifications to elucidate the activity loss from these plots are 0.9G rnin.-l for “active sites” of enzymes sometimes has resulted in the “rate” assay and 0.38 min.-’ for the “all-orthe designation of a residue as “essential to enzyme none” assay. The striking difference between activity.” I n most of these investigations, how- these two plots can be explained in terms of an ever, a decrease in activity by a factor of twenty activity dependence on two amino acid residues, would have been interpreted as “inactivation.” X and Y , independently oxidized a t rates involving I t would seem desirable, therefore, to describe the constants 0.3s min.-’ and about 0.6 min.-1, such residues as “essential to native enzyme respectively. If oxidation of X reduced enzyme activity” and to attempt to quantitate the de- activity by a factor of >200 while oxidation of Y crease in activity caused by the modification in affected activity to a considerably smaller extent, question. Accordingly different types of activity the “all-or-none” assay would be sensitive to oxiassays have been developed and tested in connection dation of X while the rate assay would be sensitive with methylene blue catalyzed photooxidation of to oxidation of both residues. This explanation could be tested by an extension the enzyme phosphoglucomutase. One of these was the conventional activity assay of the “rate” assay. The correlation between depending on the rate of the catalyzed glucose-l- residues X and U and activity by rate assay is phosphate (G-1-P) to glucose-6-phosphate (G-6-P) expressed mathematically in equation 1. Here conversion. This assay measures an average efA / A 0 = e - ( k x + k y ) t + F ( e - k x r - e-(kx+ky)$) (1) (10) H. R. Bolliger and D. A. Prins, Helu. Chin. A d a , 28, 465
Vol. 82
COMMUNICATIONS TO THE EDITOR
4740 .
.
.
.08
Both the comparison of the “rate” and “all-ornone” assays and the extension of the rate assay to large values of t lead to the same conclusion, that photooxidation of a single accessible histidine residue reduces pbsphoglucomutase activity by a factor of about 1 2 Moreover, this concordance suggests that these kinetic methods, which are not theoretically limited to phosphoglucomutase or photooxidations may be useful in correlating structure with astiv.ity in other systems.
.06
n R 0 O R H A V E N S A T I O N A I , LADORATORY
1.00
.80
.60 .40 INTERCE
.I
0
WILLIAMJ. RAY,JR. UPTON,NEWYORX J O H N J. RUSCICA T m ROCKEFELLER INSTITUTE U. E. KOSKLAND, JR. YEW Y O R K C I T Y 21, .v. Y. RECEIVED J U L Y 8, 1960
.04
CONFIGURAnON AND OPTICAL ROTATORY DISPERSION OF ATROPISOMERIC BIARYLSI Sir :
.001
!
.“X8 j
Rotatory dispersion curves have been useful in configurational correlations of compounds amtaining asymmetric atoms.* The availability of structurally symmetrical optically active biaryls of known absolute configuration3 prompted US to secure the rotatory dispersion curves of over sixty compounds in this class. We now report the salicrit results of our investigation, which lead
\
.
.
I
2
.
..
.
--A,. 6 7 8 9 I.-:
..!
3 4 5 1011 I 2 PHOTOOXIDATION TIME IN MINUTES
13
40r 1
14
Fig. 2 . - k of phosphoglucomutase activity on extensive photo6xidation as measured by the “rate” assay.
kx and k y are the rate constants for oxidation of X and Y, and F is the fractional activity in the enzyme modification in which Y has been oxidized. The activity of the enzyme in which X has been oxidized is taken as zero, ;.e., undetectable by the present assay procedures. The equation gives a straight line on a semi-log plot only for small values of t in agreement with Fig. 1 but predicts a pronounced curvature a t large values of t . When photoijxidation was followed to 99.9% loss of enzyme activity such curvature was found as shown in Fig. 2. hlorcover the appropriate graphical analysis led to the evaluation of k y , kx and F as 0.64 iiiin.-l, 0.37 min.-’ and 0.078, respectively. To identify the residue, Y, rate constants for oxidation of the various susceptible residues of the enzyrnc were determined. The first-order rate constant for oxidation of the accessible histidines was found to be 0.65 min.-I. As noted previously’ part of the histidine residues of this cnzynic are relatively inaccessible to photoKxidation (k = 0.048 min.-l).* Since the oxidation constants for other susceptible residues were considerably less than 0.04 (see Ray, ct it seems quite certain that Y can be idc~itificdas an uccessible histidine residue. (1) D . E. KosNarrd, Jr.. W . J . Kay, J r . . and M . J. Erwin. Fidrrafion P i a . , 17, 1115 (1968) (2) The rate constants arc quite dependent on the detailed rcaolinn conditions which will be described in the full publication. In general the pboto6sidations were similar to those of Weil. James and Ruchert. Arch. Bistbcm. Biopkyr.. 46, 266 (1958), except for the use of mu&
higher light /ntansities. (3) W. J. Ray, J r . , I i G . Latham. Jr , hf. Katsoulis and D. E. Koshlnnd. J r . , THIS J O I J R N A I . , a, 47.43 (1960).
25 0’
0
20
(R)-ll
(S)-Vl 0
t
(R)-l
’0
(R)-lll
(S)-V
.I
15
(S)-lV
-- - - -_ _ _ -
-.-.-._
-.-.-.-
20 300
400
500
600
700
&(my).
Fig. 1. Optical rotatory dispersion curves (dioxane) of: ( K ) - I ( S = C O ) ; ( K ) - 1 1 ( S = CHOCOCOCsII,);( K ) - I 1 1 (X = CHOII); ( S ) - R ( S C(C0zCzHb)z); (S)-V ( X = 0 ) ; (S)-VI (X = direct boud between CHZgroups).
-
( 1 ) Configurationd S t u d h in tbe Siphenyl Series. I X (paper V I I I , K . Mislow and P. A. Gr-ann, J . Or#. Chrm.. ¶S, 2027 (1968))
and ”Optical Rotatory Dispersion Studies. X X X I X ” (paper X X X V I I I , C. Djerassi, W. D. Ollis and R . C. Russell,J . Chem. Soc.. in pur). (2) C . D j e r d , “Optical Rotatory Dispersion.” McCraw- Hill Book Co.. Inc.. New Ymk, 1960; see espedally Chapter 10. (3) K. Mislow. Anpcw. Chem.. TO, 683 (1958),and references cited tbcrein.
