The effect of calcium chloride on several α-chymotrypsin-catalyzed hydrolyses

I44

BIOCHIMICA ET BIOPHYSICA ACTA

VOL. 2 8

(1958)

T H E E F F E C T O F C A L C I U M C H L O R I D E ON S E V E R A L o~-CHYMOTRYPSIN-CATALYZED HYDROLYSES ROBERT

R. J E N N I N G S ,

RICHARD

J. K E R R

AND C A R L N I E M A N N

Gates and Crellin Laboratories o/ Chemistry*, Cali/ornia Institute o/ Technology, Pasadena, Cali/. (U,S.A.)

In 195o JANDORF1 observed that the addition of magnesium sulfate caused a marked increase in the rate of the a-chymotrypsin-catalyzed hydrolysis of aeetyl-L-tyrosine ethyl ester and in 1952 SHINE AND NIEMANN2 extended the above observation to include an amide type of specific substrate, i.e., chloroacetyl-L-tyrosinamide. At approximately the same time NEURATH et al. 3, using acetyl-L-tyrosine ethyl ester and unspecified amide-type specific substrates, reported that only Ca ++ produced significant activation. It was also stated 3 that with the above ester, in systems 0.005 M in a THAM*-HC1 buffer, a maximum activation of about 50% was attained at IO 2 M C a ++, and about one-half of this amount at 2-10 -4 M. More recently W u AND LASKOWSKI4 examined the effect of calcium chloride upon reactions catalyzed by both a- and fl-chymotrypsin and, with L-phenylalanine ethyl ester as the specific substrate, obtained an extent of activation comparable to that reported by NEURATH el al. a. In a study completed several years ago '~ it was observed that the addition of calcium chloride caused a significant increase in the rate of the a-chymotrypsincatalyzed hydrolysis of L-tyrosinhydroxamide, in a system o.I M in the cacodylic acid component of a cacodylic acid sodium cacodylate buffer, that did not appear to arise from an increase in the ionic strength of the reaction system (Table I) and in this sense was distinguishable from the increase in rate associated with the addition of sodium or potassium chloride to a system containing a-chymotrypsin and ehloroacetyl-L-tyrosinamid(¢. The lesser increase in activity observed with L-tyrosinhydroxamide (Table I), relative to that reported for acetyl-L-tyrosine ethyl ester a and I.-phenylalanine ethyl ester ~ could have arisen either from a dependency of the extent of activation by calcium chloride upon the nature of the specific substrate or upon the nature and/or concentration of the buffer components of the reaction systems. There was no indication that the extent of activation by calcium chloride was pH-dependent between pH 6.0 and 7.0. Furthermore, in view of the earlier experience with magnesium sulfateL2 the data given in Table I indicated that the nature of the anionic component of the added salt m a y be of some consequence. From a second set of experiments, conducted with acetyl-L-tyrosinhydroxamide in aqueous solutions at 25 ° and pH 7.6 and 0. 3 M in the THAM component of a THAM-HC1 buffer and in the absence and presence of 0.04 ~tlr calcium chloride, o/. * ( ' o n t r i b u t i o n No. 2268. ** T r i s - ( h y d r o x y m e t h y l ) - a m i n o m e t h a n e .

YCeleren~es t9. -r47.

VOL. 28 (1958)

a-CHYMOTRYPSIN-CATALYZED HYDROLYSES TABLE

145

I

EFFECT OF SODIUM, MAGNESIUM AND CALCIUM CHLORIDE ON THE a-CHYMOTRYPSIN-CATALYZED HYDROLYSIS OF L-TYROSINHYDROXAMIDE L-Tyrosinhydroxarnide in aqueous solutions at 25 ° with [E] = o.2o8 mg protein-nitrogen/ml of A r m o u r p r e p a r a t i o n N o . 9 0 , 4 0 2 a n d [S]o = i o . lO -3 M ; p H t o w i t h i n =L o . i o f a p H u n i t ; b u f f e r : o . i M i n t h e c a c o d y l i c a c i d c o m p o n e n t o f a c a c o d y l i c a c i d - s o d i u m c a c o d y l a t e b u f f e r ; [S]o/[S]t i n u n i t s o f lO -3 r a i n - 1 .

pH

Added component

7.o 7.o 7.0 6.0 6.0 6.0 6.0

None o.o2 M MgCI 3 0 . 0 2 M CaC13 None o . o 6 M NaC12 o . o 2 M MgC12 o . o 2 M CaC13

Ionic strength Added comp. -o.o6 0.06 -o.o6 o.o6 o.o6

Buffer

I/6o × Total

M([S]°/[S]t)

o.o9 o.15 o.15 0.05 o.Io O.lO o.Io

15.5 16. 4 18. 3 5.39 5.5 o

o.o9 o.o9 o.o9 0.05 o.o 5 o.o 5 o.o 5

TABLE

Relative activity

I.OO 1.o6 1.18 I.OO 1.o2 I.OO 1.17

5.41 6.33

II

EFFECT OF CALCIUM CHLORIDE ON THE a-CHYMOTRYPSIN-CATALYZED HYDROLYSIS OF ACETYL-L-TYROSINHYDROXAMIDE L - T y r o s i n h y d r o x a m i d e i n a q u e o u s s o l u t i o n s a t 25 ° a n d p H 7 . 6 ± o . I a n d o . 3 M i n t h e T H A M c o m ponent of a THAM-HC1 buffer with [E] = o.o40 mg protein-nitrogen/ml of Armour preparation N o . i o , 7 o 5 ; [S]0 i n u n i t s o f lO -3 M ; v 0 i n u n i t s o f lO -5 :&!/min; [S]o/v o i n m i n u t e s .

[S]o 3.00 IO.O io.o 3°.0 50.0 70.0

Vo 8.77 25.2 24. 3 55-5 72.0 86. 3

[S]o/Vo 34.2 39.6 41.I 5 4 .0 69.5 81.o

[S]o

Vo

[S]o/vo

0 . 0 4 M CaC12 5.21 16.79 39.84 7o.44

18.o 47.5 82.0 ioi.o

29.o 35.2 48.5 69.6

T a b l e I I , a s u b j e c t i v e e s t i m a t e of t h e slopes a n d i n t e r c e p t s of t h e two v o vs. [S]o/V o plots led to values of Ks* of 45 a n d 4 ° a n d k a * of 35 a n d 4 ° . T h e values of Ks a n d k3 o b s e r v e d in t h e absence of a d d e d calcium chloride are in r e a s o n a b l e a g r e e m e n t w i t h those o b t a i n e d p r e v i o u s l y 7, i.e., 43 :k 4 a n d 33 zL 3- Those o b t a i n e d in the presence of calcium chloride suggest t h a t K s is r e l a t i v e l y insensitive to the presence of calcium chloride a n d t h a t the value of k 3 m a y be g r e a t e r t h a n in t h e absence of this salt. H o w e v e r , from t h e r a t i o s of k3/Ks it follows t h a t 0.04 M calcium chloride led to an e x p e c t e d increase in r a t e of 28 % when b o t h r e a c t i o n s are e x a m i n e d u n d e r c o n d i t i o n s where t h e y are a p p a r e n t first o r d e r w i t h respect to specific s u b s t r a t e . I n o r d e r to o b t a i n a d d i t i o n a l i n f o r m a t i o n of the d e p e n d e n c e of t h e e x t e n t of a c t i v a t i o n a s s o c i a t e d w i t h t h e presence of calcium chloride u p o n the c o n c e n t r a t i o n of t h e buffer c o m p o n e n t s of t h e r e a c t i o n s y s t e m a series of e x p e r i m e n t s were c o n d u c t e d w i t h a - N - n i c o t i n y l - L - t y r o s i n h y d r a z i d e u n d e r conditions where the buffer concent r a t i o n Was m a i n t a i n e d a t o.o2 a n d I.O M in the T H A M c o m p o n e n t of a T H A M - H C 1 buffer a n d the calcium chloride c o n c e n t r a t i o n was v a r i e d over a I O to lO 5 fold range, * A l l v a l u e s o f K S a r e i n u n i t s o f IO - 3 2~I a n d t h o s e o f k a i n u n i t s o f . I o -3 M / m i n / m g nitrogen/ml.

Re/erences p. z47.

protein-

146

VOL. 28 (1958)

R . R . JENNINGS, R. J. KERR, C. NIEMANN

c/. T a b l e I I I . T h e i n i t i a l v e l o c i t i e s w e r e e v a l u a t e d b y t h e m e t h o d of JENNINGS AND ~qlEMANN s a n d b y t h e e m p i r i c a l o r t h o g o n a l p o l y n o m i a l p r o c e d u r e of BOOMAN AND NIEMANN 9. TABLE III EFFECT OF CALCIUM CHLORIDE AND OF V E E S E N E ON THE a-CHYMOTRYPSIN-CATALYZED HYDROLYSIS OF ¢2-N-NICOTINYL-L-TYROSINHYDRAZIDE

Versene: Disodium hydrogen salt of ethylenetetraacetic acid; a-N-nicotyl-e-tyrosinhydrazide in aqueous solution at 25 ° and p H 7.9 ± o.i with [E] = o.218 mg protein-nitrogen/ml of Armour preparation No. 00592 and [S] 0 = 1 .DO3. IO-3 M ; buffer: M of the THAM component of a T H A M HC1 buffer; vo in units of lO.5 M / m i n evaluated by the method of J E N N I N G S AND N I E M A N N 8 ; v' 0 in units of lO-5 M/rain, evaluated by the procedure of BOOMAN AND N I E M A N N g ; P r o : order of polynomial used for fitting of nine points observed for maximum time of reaction of 9 or IO rain corresponding to extents of reaction of from 22.0 to 44.6 %. (Buffer)

(CaCl,)

o.o2

o o lO-6 lO-5 lO -4 lO-3 IO-Z IO-1

2.62 2-67 2.53 2.68 3.15 4.24 4.35 4.63

2.95 2.45 2.42 2.49 3.32 4.3 ° 4.52 4.45

-- o.22 ~: o,27 :L o.27 4- o.28 ~ o.18 ! o.15 :J: 0.25 i 0-56

3 3 3 3 2 3 3 2

I.OO

o lO-5 lO-4 lO-3 IO-z

3-80 4.20 4.42 5 .10 5.32

3.63 4.18 4.49 4.97 5.32

:t: 4± + :j-

o.18 0.26 0.34 o.41 0.52

3 3 3 2 2

(Versene) M o 5.3" IO-6 8.9" IO-6 15.1" IO-e

2.7 ° 2.58 2.66 2.72

2.51 2.43 2.55 2.67

± ± ~±

o.39 0.08 0.20 0.26

3 2 2 2

M

0.02

M

Vo

v" o

Pm

I t will b e s e e n f r o m t h e d a t a s u m m a r i z e d i n T a b l e I I I t h a t i n 0.02 M T H A M significant activation by calcium chloride begins to appear at concentrations between 10 -5 a n d 10 -4 M , r e a c h e s a n a p p a r e n t m a x i m u m , w h i c h i n r e a l i t y is a p l a t e a u , b e t w e e n 10 -3 a n d 10 -3 M a n d t h e n c o n t i n u e s t o i n c r e a s e a t c o n c e n t r a t i o n s a b o v e 10 -2 M . T h e e x t e n t o f a c t i v a t i o n a t 10 -3 t o 10 -3 M c a l c i u m c h l o r i d e , i.e. a b o u t 6 0 % , is c o m p a r a b l e t o t h a t o b s e r v e d p r e v i o u s l y b y NEURATH et al. 3 a n d b y W o AND LAWNOWSKI4 f o r s y s t e m s o f r e l a t i v e l y l o w i o n i c s t r e n g t h . I n 1.0 M T H A M a c t i v a t i o n b y c a l c i u m c h l o r i d e is s e e n a t 10 -5 M a n d c o n t i n u e s t o i n c r e a s e , w i t h n o e v i d e n c e for a n y m a r k e d discontinuities, w i t h increasing c o n c e n t r a t i o n of the a d d e d salt. H o w e v e r , t h e e x t e n t o f a c t i v a t i o n a t t a i n e d b y t h e a d d i t i o n o f I 0 -~ M c a l c i u m c h l o r i d e i n t h e l a t t e r c a s e is b u t a b o u t t w o - t h i r d s of t h a t o b s e r v e d a t t h e l o w e r b u f f e r c o n c e n t r a t i o n . I t a p p e a r s t h a t t h e m a r k e d rise i n a c t i v i t y t h a t o c c u r s b e t w e e n 10 -4 a n d I 0 -a M c a l c i u m c h l o r i d e i n t h e 0.02 M T H A M b u f f e r is l o s t a t l e a s t i n p a r t i n s y s t e m s of h i g h e r buffer c o n c e n t r a t i o n . T h e fact t h a t t h e rates in t h e a b s e n c e a n d p r e s e n c e

Re[erences p. 147.

VOL. 9.8 (1958)

a-CHYMOTRYPSIN-CATALYZED HYDROLYSES

147

of Versene are identical with the limits of experimental error, c/. Table III, is consistent with the earlier conclusion of NEURATH et al. 3, based upon spectroscopic evidence, that a-chymotrypsin is substantially free of calcium salts. I t is clear that the dependence of the extent of activation associated with the presence of calcium chloride upon the nature and/or concentration of the buffer components of the reaction system, which has been demonstrated above, explains in part the discrepancies noted earlier in this communication. However, in view of this dependence and an additional possible dependence upon the nature of the specific substrate it follows that an interpretation of the nature of the apparent activation of a-chymotrypsin-catalyzed hydrolyses b y calcium chloride must be deferred until further information is available with respect to the behavior of several specific substrates in systems of varying enzyme concentration under conditions where the systems are maintained at a constant p H but in the absence of conventional buffers. EXPERIMENTAL

The experiments involving L-tyrosinhydroxamide and acetyl-L-tyrosinhydroxamide were conducted essentially as described b y HOGNESS AND NIEMANN1° and FOSTER, JENNINGS AND NIEMANNn. Those with a-N-nicotinyl-L-tyrosinhydrazide as described b y LUTWACK, MOWER AND NIEMANN 12 a n d K E R R AND NIEMANN 1~. SUMMARY T h e e x t e n t t o w h i c h t h e r e a c t i v i t y of s y s t e m s containing a - c h y m o t r y p s i n a n d specific s u b s t r a t e s of this e n z y m e m a y be e n h a n c e d b y t h e presence of calcium chloride h a s been s h o w n to be dep e n d e n t n o t only u p o n t h e c o n c e n t r a t i o n of this salt b u t also u p o n t h e n a t u r e a n d / o r c o n c e n t r a t i o n of several buffer c o m p o n e n t s . A n additional dependence u p o n the n a t u r e of t h e specific s u b s t r a t e h a s n o t been excluded.

REFERENCES 1 B. J. JANDORF, Federation Proc., 9 (195 o) 186. 3 H . J. SHINE AND C. NIEMANN, J. Am. Chem. Sot., 74 (I952) 97. 3 M. M. GREEN, J. A. GLADNER, L. W. CUNNINGHAM, JR. AND H. NEUEATH, J. Am. Chem. Soc. 74 (1952 ) 2122. 4 F. C. W u AND i . LASKOWSKI, Biochim. Biophys. Acta, 19 (1956) IiO. 5 R. R. JENNINGS, P h . D . Thesis, Calif. I n s t . Techn., Pasadena, Calif., 1955. e H. J. SHINE AND C. NIEMANN, J. Am. Chem. Soc., 77 (1955) 4275 • 7 H. J. FOSTER AND C. NIEMANN, J. Am. Chem. Soc., 77 (1955) 1886. 8 R. R. JENNINGS AND C. NIEMANN, J. Am. Chem. Soc., 75 (1953) 4687 • 3 K. A: BOOMAN AND C. NIEMANN, J. Am. Chem. Soc., 78 (1956) 3642. lO D. S. HOCrNESS AND C. NIEMANN, J. Am. Chem. Sot., 75 (1953) 884. 11 R. J. FOSTER, R. R. JENNINGS AND C. NIEMANN, J. Am. Chem. Soc., 76 (1954) 3142. x3 R. LOTWACK, H. F. MOWER AND C. NIEMANN, J. Am. Chem. Soc., 79 (1957) 2179. 13 1R. J. KERR AND C. NIEMANN, J. Am. Chem. Soc., in the press.

Received October i6th, 1957