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THE JOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 248, No. 5,Issue of March 10, pp. 1741-1745, 1973


Printed in U.S.A.

Asparaginase
STEREOCHEMISTRY OF THE ACTIVE CESTER AS DETERMINED BY CIRCULAR DICHROISXI OF
SUBSTRATES*

(Received for publicat,ion, July 7, 1972)

JOSEPH E. COLEMAN AND ROBERT E. ~ANDscHunIAcHER


From the Departments o;f Molecular Biophysics and Biochemistry and Pharmacology, Yale University,
New Haven, Connecticut 06510

SUMMARY broad range of activity has been extended t’o substrates contain-
Large Cotton effects associated with the r + T* and ing 2 asymmetrical carbon atoms which show large changes in
n + g* transitions of asparaginase substrates have been optical activity accompanying hydrolysis of the amide. From
shown in substrates containing a 2nd asymmetrical carbon the changes in the substrate Cotton effect during hydrolysis it

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atom at the p position, diaminosuccinic acid monoamide and has been possible to define the absolute stereochemical require-
ments of this enzyme in positions 2 and 3 of the substrate.
@-methylasparagine. Hydrolysis of the amide bond is ac-
companied by large changes in the rotatory strength of these
transitions. These substrates have four possible isomers, MATERIALS AND METHODS

L-L, D-D, L-D, and D-L. From the signs of the Cotton effects
Xubstrates and Enzymes-L-Aspartic acid, L-asparagirre, and L-
appearing on hydrolysis of mixtures of these isomers it can leucine were chromatographically pure (Schwarz-Mann, New
be determined which isomer is hydrolyzed. The enzyme York). L-Leucine amide was a gift of Professor Sofia Simmonds
has an almost absolute stereospecificity for the L-D isomer
of Yale University. D-L, L-n-Diaminosuccinic acid monoamide
which defines the three-dimensional arrangement of the and L-L, D-D-diaminosuccinic acid monoamide were synthesized
protein groups interacting with the substituents on the o(
by Dr. Pauline Chang, Yale University, and the syntheses will
carbon and the catalytic groups adjacent to the P-amide
be reported in detail elsewhere (9). P-Methylasparagine (L-L,
within rather narrow limits. While the L-L isomer of D-D) was also synthesized by Dr. Chang starting with commercial
diaminosuccinic acid monoamide is not detectably hydro-
three-DL-fi-methylaspartic acid (Nutritional Biochemicals, Cleve-
lyzed, the L-L isomer of P-methylasparagine is attacked at a
land, 0.). Asparaginase (270 units per mg) from E. coli B was
rate 0.2 to 0.3 % of that observed for L-asparagine, suggesting prepared by Merck and Co. and supplied as a lyophilized sample
that a P-CH3 group can be accommodated in this configura-
of an electrophoretically homogeneous preparation. One unit
tion while a ,&NH2 cannot. of asparaginase is the amount of protein required for the release
of 1 pmole of ammonia per min from L-asparagine at pH 8.5, 37”.
Optical Rotatory Dispersion and Circular Dichroismn-ORDr
was recorded with a Cary 60 recording spectropolarimeter. CD
leas recorded with a Cary 61 spcctropolarimeter. Slit widths
The enzyme L-asparaginase (L-asparagine amidohydrolase, were programmed to maintain constant light intensity. The CD
EC 3.5.1.1) has been the object of increasing interest since the instrument was calibrated with an aqueou:: solution of d-10-
purified enzyme has been found useful in the treatment of acute camphorsulfonic acid (J. T. Baker Co.). en - Ed = 2.20 at 290
lymphoblastic leukemia (l-4). Its clinical utility is attributed nm. All measurements were carried out at 25”. ORD is ex-
to the asparagine-dependent nature of these neoplastic cells in pressed as specific rotation, [a], or as molecular rotation, [M] =
contrast to normal cells which can synthesize asparagine (4). [a]M.W./lOO, in units of dcg cm2 per dccimole. CD is espressed
The physicochemical properties of the crystalline enzymes from
in terms of molecular cllipticity, [e] = 2.303 (45OO:l7r) (EL - en)
Escherichia coli B and Erwinia caratovora have been reported on
wit’h units of dcg cm2 per decimole.
extensively (5-7) _ The protein is a tetramer of molecular Tveight
-133,000 (4, 5, 7).
RESULTS
A variety of alternate substrates for the enzyme have been de-
scribed including /3-cyanoalanine and the P-hydroxamate of L- Substrate and Optical Properties-The primary substrate em-
aspartic acid both of which are converted to L-aspartic acid (4). ployed iu this study was the monoamide of diaminosuccinic acid
Catalytic decomposition of 5-diazo-4-oxo-L-norvaline as well as (Scheme I). Since there are 2 asymmetrical carbon atoms the
active site labeling by this reagent have been reported (8). This compound can exist as four different stereoisomers,
* This work was supported by Grants AM-09070-08 and CA-
10748 from the National Institutes of Health and Grants IC-64L 1 The abbreviations IIsed are: ORD, optical rotatory dispersion;
and PR-27 from the American Cancer Societv. CD, circular dichroism.

1741
1742

0 NH,H 0
Ii I I II
-o-c-c-c-c
I I \
H NH2 NH,

D L
or
I, D

SCHEME I
LL, nn, LD, and DL. For convenience in comparing this corn
pound to asparagine the carbon adjacent to the carboxyl is desig-
nated as the (Y carbon and that carrying the amide as the fi car-
bon. The parent compound, diaminosuccinic acid exists as three
stercoisomcrs LL, DD, and the meso form (DL = LD). Since the
first two isomers can be readily separated from the meso form, the
meso form of diaminosuccinic acid has been used for synthesis of 13
the amide substrate. The product should contain two meso-type
isomers of diaminosuccinic acid monoamide, ~-L-P-D and (Y-D-@-L 4
(Scheme I). The product of this synthesis is not optically active

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(Fig. 1) confirming that the product must contain either tlvo i 5
6
isomers showing equal and opposite rotations, or a single isomer
with two asymmetrical centers (of opposite configuration) which
make equal contributions to the rotation. Since the amide 210 220 230 240 250 260
x, nm
chromophore will not make the same contribution to rotation as
the carboxyl chromophore (see below), the first alternative ap- FIG. 1. ORI1 (CWW 1) and CD (Curve 2) spectra of asolution of
~-D-&I- and LY-r,-P-D-diiLmirlosI1ccinic acid monoamide (1.84 X
pears more likely and suggests the presence of the two isomers in IOP RI) after 24 hollrs incrlbation with 10 ~1 of nsparaqinase (0.164
equal concentrations as expected from the synthesis. Since mg per ml). The WTOT bars on the axis indicate the ranges of a
hydrolysis of only one of the isomers is expected to be catalyzed base-line Cl> sprctrmn taken on the same solution untreated with
by the enzyme, complete enzymatic hydrolysis should be ac- enzyme. Condilions: 0.05 M Tris, pH 8.0, 25”, 0.2.cm path length.
compallied by the appearance of optical activity at the wave
length of the absorption bands of the unhydrolyzed amide. A enzyme activity. l-llfortunately in the caec of the mixture of
solution of the diaminosuccinic acid amide treated with asparagi- a-L-&D- and ol-1)-P-L-diamirlosuccinic acid monoamide the1 c is
nase for 24 hours shows a large negative Cotton effect centered severe subst~rate inhibition at substrate concentrations >5 x
near 222 nm (Fig. 1). This suggests that only 50% of the sub- 1OW X. This inhibition prevented a complete kinetic analysis.
strate is hydrolyzed as has been confirmed by determining am- The maximum turnover observed for diaminosuccinic acid mono-
monia release by Nesslerization (9). Enzymatic hydrolysis re- amide n-as 4.75 X 10” moles of substrate hydrolyzed per min per
sults in the release of only 50% as much ammonia nitrogen as does molt of enzyme in 0.05 nr Tris .HCI, pH 8.0, at a substrate concen-
complete acid hydrolysis of the substrate (9). Once maximum tration of 1 X 1OP nr. This number is the same order of nlagni-
rotation is achieved the CD spectrum remains the same indefi- tude as the turnover lrumbcrs reported for L-asl)umgine as sub-
nitely suggesting that the other isomer is completely resistant to strate under similar conditions (4, 5). The highest maximum
hydrolysis. velocity rcportcd for t,he hydrolysis of L-asparagine is 8.95 x 104
Rate of Change of Ellipticity as Function of Enzyme Concentra- moles per min per mole of enzyme, as assayed by the coupled
tion-Enzymatic hydrolysis of 50% of the mixture of mesoiso- spectrophotomctric assay (5). The substrate inhibition may re-
mers of diaminosuccinic acid monoamide yields a product which late to the prcscncc of the unhydrolyzcd isomer and it is possible
has a combined molar ellipt’icity at the minimum of the CD that with a pure isomer this CD assay could be devclopcd into one
spectrum of -6.3 X lo2 deg cm2 per decimole (Fig. 1). Com- satisfactory for a complet~c kinetic analysis. It would have the
plete enzymatic hydrolysis of a 6.85 mM solution (1 mg per ml) advantages of a direct spcctrophotometric assay without the
observed over a l-cm path yields a change in observed ellipticity addition of other enzymes as in the assay coupled to the reduction
of -0.0445” at 222 nm. Appearance of the 222.nm Cotton effect of pyridine nucltotidc (1 I) or the analytical problems of mcnsur-
is directly related to enzymatic hydrolysis of the substrate, since ing ammonia release.
the rate of appearance of this Cotton effect is directly propor- Stereochemistry of Isomer of Diaminosuccinic Acid Monoanzide
tional t’o enzyme concentration (Fig. 2). Time courses for the Hydrolyzed by Aspclraginase-The Cotton effect gcncrated b>
increase in negative ellipticity at 222 nm at three different en- 50 y0 hydrolysis of the misturc of mcsoisomers of diaminosuccinic
zyme concentrations are shown. The rate function over a 6-fold acid monoamidc is negative (Fig. I). Deduction of the sterro-
range of enzyme concentration is shown in the inset to Fig. 2. chemistry of the hydrolyzed and unhydrolyzed isomers from the
The pen period on the Cary 61 was set at 1 s to insure rapid sign of this Cotton cffcct requires both an assignment of this
pen response. The resulting large random noise does not, how- transition and a knomlcdge of its sign in cnvironmcnts of known
ever, reduce the precision of the slope over that of determina- symmetry. This can readily be deduced from the CD of some
tions taken at longer pen periods with less random noise. Thus model compounds. Aliphatic amino acids of the L configuration
the change in cllipticity can be adapted as a satisfactory assay of iucluding r,-aspnrtic and L-glutamic acids have positive Cotton
1743

effects near 200 nm due largely to the overlapping r + 7r* and n strength and the low wave length of the change near 210 nm,
+ r* transitions of the carboql chromophore at the cy posi- make it unsatisfactory to follow asparagine hydrolysis wit.h op-
tion (Fig. 3). The n + r* transitiou of the cnrbosyl group is tical rotation (Fig. 3). However, if an amide is substituted for
usually near 204 nm (10). If the p- or y-carboxyl is changed to the cu-carboxyl, the n ---) T* transition shifts to the region of
an amide chromophore as in L-asparagine there is a small but sig- 220 nm with a resultant change in the CD pattern in the region
nificant change in the rotation (Fig. 3). The change in clliptic- of 220 nm. This is illustrated in Fig. 4 by the CD spectra of
ity from the amide to the acid is relatively small, since it is due to L-leucine and L-leucine amide. The free acid shows the typi-
the vicinal effect of the asymmetrical center locat’ed 2 carbon cal positive Cotton effect at 198 nm, but a prominent negaOive
atoms away. The relatively small change in optical rotatory band appears at 227 nm in the amide. The latter can be as-
signed to the n ---) 7r* transition which undergoes a pronounced
red shift in amides (10). The n --j P* Cotton effect is negative
when the amide is adjacent to a carbon in the L configuration.
Thus in the case of the diaminosuccinic acid monoamide hydro-
lyzed by asparaginase, the Cotton effect at 222 nm is due t,o the
unhydrolyzcd amide and that amide is of the L configuration
(Fig. l), hence the L-D isomer must be the one hydrolyzed. The
relationship of this finding to the stereochemistry of the active
site of the enzyme will be discussed below (SW discussion).
If a mixture of ~-L-,&L- and a-D-fl-n-diaminosuccinic acid mon-
oamide ‘2HBr (2.7 mg per ml) is treated with up to 0.10 mgperml
of asparaginase no optical rotatory changes are observed over a
period of 72 hours. Furthermore, t’he enzyme did not release a

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significant amount of ammonia from the mixture of C&L and
a~-/?D isomers over this time period.
Hydrolysis of ,L-Methylasparagine-Although no hydrolysis of
the a-~-p-~ and (Y-D-@-D mixture of diaminosuccinic acid amide
was detected, a very slow hydrolysis of the ~-L-P-L and ~-D&D
isomers of fl-methylasparagine was observed. This mixture of
isomers can be prepared by amidation of the commercially avail-
able three-nL&methylaspartic acid. The Cotton effects appear-
200 100
TIME, 5cc ing upon treating a mixture of (Y-L-~-L- and ac-D-fl-D-P-methyl-
FIG. 2. Changes in observed ellipticity, Bobs, at 222 nm as a asparagine with asparaginase are shown in Fig. 58. A large
function of time afler additions of 0.85, 1.76, and 2.64 units of negative ellipticity band appears at 204 nm and an apparent
nsyaraginasc (0.164 mg per ml) to a solution of WI-B-D-, a-~-p-~- smaller negative elipticity band at ~220 nm (Fig. 5A).
diaminosuccinic acid monoamide.2HBr (4.8 X 1OP M) contained
in 0.05 M Tris, pH 8.0, 25”. Inset, Bobs per min as a function of I I\ I
asparaginasc concentration, 0.44 unit/l0 ~1. To insure adequate I
pen response the time constant on the Gary 61 was set at I s. - 0
c-o-
0
--- II
C-NH2
+3

n
b- I
x
G

0
x5
t
227

-I

200 225 250 1 I I I I I I I I I L


X,nm 200 225 2 50
X,nm
IGo. 3. CD spectra of L-asparagine (- - -), L-xspartic acid
(--), and L-aspnragine after complete hydrolysis by asparaginase FIG. 4. CD spectra of L-leucine (---) and L-leucine amide
(....). Conditions: 0.05 M Tris, 3.75 X 10M3 M amino acid, 25”, (---). Conditions: 0.05 M Tris, 3.S X 10-a M amino acid, 25”,
0.2-cm path. 0.2-cm path.
1744

At the enzyme concentration required to observe significant due to the enzyme. In the case of fl-methylasparagine no clear
hydrolysis, holr-ever, the enzyme itself makes a significant contri- n * @ amide transition is separated out between 220 and 230 nm
bution to the CD spectrum and the ellipticity at 220 nm has a as in the cnsc of the diaminosuccinic arid amide (Fig. 1). Thus
large contribution from the enzyme. Curve 1, Fig. 58, is largely in order to be certain of the stereochemistry of the isomer hy-
drolyzed, it was necessary to separate the product acid from the
I 1 I I I I I I unhydrolyzed amide. This was accomplished with a Dowes I-
X2 column and the CD spectra of the resolved compounds are
A shown in Fig. 5B. The unhydrolyzed amide is of the WD+-D con-
figuration, giving rise to large negative Cotton effects, while the
hydrolyzed isomer is of the cy-L-~-L configuration, giving rise to
large positive Cotton effects. The net increase in negative ellip-
ticity observed on hydrolysis (Fig. 5A) is due to a decrease in
magnitude and slight blue shift of the positive Cotton effects for
the a-L-&L-acid compared to the oc-r-/-L-amide. These shifts
in CD between the acid and the amide are more like those occur-
ring on hydrolysis of asparagine (Fig. 2) where no clear separation
of the 7r + QT* and n + r* transitions is present,
The ~-L-&L isomer of P-methylasparagine is hydrolyzed only
0.3% as fast as the a-L-,&r-diaminosuccinic acid monoamide as
shown by the time course for dcvclopment of the Cotton effects
in Fig. 5A. The turnover number calculated from this data is
only 150 moles of substrate hydrolyzed per min per mole of en-

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1.50 zyme. This is significantly faster, however, than the hydrolysis
of the ~-L-,&L isomer of diaminosuccinic acid amide.

i DISCUSSION
200 225 250
The bond hydrolyzed in the case of the natural substrate for
X,nm
asparaginase is part of a chromophore located 2 carbon atoms
1 I I I I I I away from the asymmetrical center, and hence the rotatory
changes accompanying this hydrolysis are qualitatively less sig-
PRODUCT ACID nificant than if the amide bond hydrolyzed involved a carbon ad-
6 L-L B -methyl aspartic acid jacent to an asymmetrical center (Figs. 3 and 4). The diamino-
\
\ succinic acid derivative maintains the substrate requirements for
\
\
\ asparaginasc, but introduces an asymmetrical carbon adjacent
\ to the carbon of the hydrolyzed amide bond. This greatly en-
.
‘: 0
o-
hances t,he rotatory change near 225 nm on hydrolysis as well as
x moving the change to the more specific n ---) rr* transition of the
amide (Fig. 1). Such a procedure may be applicable to the sub-
s strates of other enzymes where the development of assays based
-6 on change in optical activity are desirable.
UNHYDROLYZED AMIDE Since the natural substrate for the enzyme is L-asparagine and
D-D B-methyl asparagine D-asparagine is poorly hydrolyzed (4, II), the proper relationship
between t’he enzyme-binding groups of the substrate and the
-12 catalytic centers on the enzyme resulting in the hydrolysis of the
6 P-amide presumably requires the L-configuration about the cz
I I I I I I I carbon. Since the p carbon is not asymmetrical the amide can
190 200 210 220 230 240 250 assume all positions of the D or L configuration of the /3 carbon.
1 , nm Whether there is a strict stereospecificity for the P-amide once
FIG. 5. A, CD changes as a function of time during the hydroly- the substrate is in place on the enzyme surface cannot be deter-
sis of a mixture of ~-L-P-L- and ol-u-p-D-(3-methylasparagine by mined.
asparaginase. Conditions: 0.05 M Tris, pH 8.0, 1.54 X IOP M In the case of diaminosuccinic acid amide, stereospecificity is
@-methylasparagine, 7.6 units (0.028 mg) if aspaiaginase per ml, conferred on the fl carbon, and thus the enzyme active site might
O.l-cm path. Curve 1 taken immediatelv after addition of the
enzyme-is largely ellipticity contributed “by the protein since at accept only one of the /3 carbon configurations. When the mix-
this concentration of enzyme the ultraviolet Cotton effects of the ture of isomers is of the oc-u-/-L-NH2 and ~~-L-P-D-NH~ configura-
protein are detected. Curve !Z is after 110 min of incubation at 25”; tions, the c~-L-P-D-NH~ configuration is hydrolyzed (Fig. I).
Curve 3 after 140 min, Curve 4 after 24 or 48 hours. The error bars This is in keeping with the analogy to asparagine where the con-
on the axis indicate the ranges ot a base-line CD spectrum taken
on the same solution untreated with enzyme. B, ultraviolet CD figuration around the carbon carrying the carboxyl must be L.
of cr-L-&L&methylaspartic acid (- - -) and a-D-p-D-p-methyl- The significance of the D configuration at the amide in the rapidly
asparagine (-). A4 mixture of a-L-~-L- and a-D-p-D-p-methyl- hydrolyzed substrate was not apparent until enzymatic hydrolysis
asparagine was treated with aspnraginase until complete hydroly- of t,he alternate pair of isomers, LY-L-P-L-NH~ and o(-D-,KD-NH~
sis had taken place (509” ammonia release). The mixture was was attempted. Ko significant hydrolysis was detected and
then applied to a column of Dowex I-X2 to separate the product
acid from the unhvdrolvzed amide. Conditions for CD as in thus the CGL-/!-L-NH~ isomer does not meet the requirements.
I .
Fig. 5A. Hence t,he catalytic region of the enzyme interacting with the
1745

relationships for t,he (Y-L-/-U, a-~-p-~ pair are illustrated in the


stereodrawing in Fig. 6.
The ~-L-/~-II isomer is shown in the extended form, since this is
the likely form in solution with the amino groups on opposite sides
of the C-2-C-3 bond. The relationship of the binding points
on the protein receiving t’he groups around the cycarbon (carboxyl
and amino) and the catalyt,ic groups (/l and U) attacking the
amide bond is then as shown by the a-~-,&n isomer in Fig. 6.
This stereochemistry is not absolutely fixed in the sense that the
configuration of the a-L-p-n isomer can change from that shown
in Fig. 6 by rotation about the a-P C--C bond. Although exten-
sive deviation from the extended form dots not appear likely, the
enzyme-substrate dissociation constant apprars to be relatively
small and binding could force some changes from the extended
configuration. Such rotation does not change the general fea-
tures of substrate-enzyme stereochemistry pictured in Fig. 6.
While the p-amino group in the C+L-P-L isomer of diaminosuc-
L-D. FORM cinic acid amide appears to interfere with the proper positioning
of the substrate, a methyl group can be accommodated in this po-
sition, although the rate of hydrolysis is much decreased over the
rate observed if a hydrogen occupies this position. Steric factors
would appear to be primarily responsible for the decreasing rate of

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hydrolysis in the order H > CHB > NH2 when these groups in the
p position are placed in the L-configuration. These steric factors
may influence the binding step or alter the relationship between
the catalytic groups and the substrate once the latter is bound.
The catalytic groups are represented in purely hypothetical
fashion as a base, B, attacking the carbonyl carbon, perhaps as-
sisted by donation of a proton from a protein group, AH, to the
amide. A specific nucleophilic attack is pictured, since data on
l*O exchange (12) and on the formation of fi-aspartohydroxamic
acid catalyzed by the enzyme (12, 13) have suggested but not
proved the formation of an acyl enzyme intermediat,c during hy-
drolysis by asparaginase.

Acknowledgments-The technical assistance of Miss Judith


FIG. 6. Stereodrawing of the conformations of the ~-L-P-D (A) Pascale and Miss Celeste 9. Gaumond is much appreciated. We
and ~-L-O-L (B) isomers of diaminosuccinic acid monoamide. The
thank Miss Phyllis Salzman for separating the isomers of fl-
hypothetical catalytic groups of the enzyme are represented as a
Base B and an acidic group AH. The isomers are both shown in methylasparagine. We are indebted to Dr. Jesse &hilling of the
an extended form with the --NH2 groups on opposite sides of the Department of Molecular Biophysics and Biochemistry for pro-
C-2-C-3 bond and occupying the same positions in the ~-L-P-D gramming the stereodrawings.
and ~-L-@-L configurations. This results in the susceptible amide
bond pointing away from the catalytic groups in the ~-L-@-L REFERENCES
isomer. 1. KIDD, J. G. (1953) J. Exp. Med. 98, 565-605
2. BROO~SE, J. D. (1961) Nature 191, 1114-1115
amide group and the site on the protein accepting the binding 3. MASHBURK, L. T., AND WRISTON, J. C. (1964) Arch. Biochem.
groups around the Q: carbon arc located in the configuration shown Biophys. 106, 451-452
by the a-L&n-isomer. Both the cu-carboxyl and a-amino groups 4. WRISTON, J. C. (1971) Enzymes 4, 101-121
5. Ho, P. P. K., MILIKIN, E. B., BOBBITT, J. L.. GRIXN.LK, E. L..
have been implicated as having binding interactions with the BURCK, P..J., FRANK, B. fK., BOECI~, L. lb)., AND SQUIRES;
protein in the case of asparaginase from E. carotovora (12). R. W. (1970) J. Biol. Chem. 246, 3708-3715
It appears that the L configuration of substituents at. the fl 6. FRANK, g. H.; PEKAR, A. H., VI&OS, A. J., AND Ho, P. P. K.
carbon interferes with the proper positioning of the substrate if (1970) J. Biol. Chem. 246, 3716-3724
the binding groups at the OLcarbon are confined to the L configura- 7. CAMMACK, K. A., MARLBOROUGH, D. I., AND MILLER, D. S.
(1972) Biochem. J. 126, 361-379
Dion. If the amide group of the (Y-L-/-L isomer is to be placed in 8. JACKSON, R.C., AND HANDSCHUMACHER, R. IZ. (1970) Biochem-
the same position as that of the effectively hydrolyzed ~-L-@-D istry 9, 3585-3590
isomer, then the p-amino group occupies the position occupied by 9. HANDSCHUMACHER, R. E., GAUMOND,~. A., AKD CHANG, P. C.
the P-hydrogen in the (Y-L-P-D isomer. This site may accommo- (1972) Biochem. kiophys. Res. Cor&zun.,.in press
10. JAFFE. H. H.. AND ORCHIN. M. (1962) Theoru and Abdications
date a hydrogen in this position but not an amino group. Xter- of havioiet Spectroscopy
~ I

p. 178, John Wiley and Sons,


‘ <

natively if binding requires that the substituents on the cx carbon New York
be in the L configuration and that the amino group of the 0 car- 11. Ho, P. P. K., AND JONES, L. (1969) Biochim. Biophys. Ada
bon be in the position it occupies in the a-~-p-~ isomer, then t’he 177, 172-174
placement of the P-amino group in this same position for the cy- 12. HOWARD, J. B., AND CARPENTER, F. H. (1972) J. Biol. Chem.
247, 1020-1030
L-@-L isomer results in rotation of the amide away from the site 13. EHRMAN, M., CEDAR, H., AND SCHWARTZ, J. H. (1971) J. Biol.
that must contain the catalytic groups on the protein. These Chem. 246, 88-94