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Asparaginase
STEREOCHEMISTRY OF THE ACTIVE CESTER AS DETERMINED BY CIRCULAR DICHROISXI OF
SUBSTRATES*
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
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
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
n
b- I
x
G
0
x5
t
227
-I
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-
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
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