The effect of γ-carboxyglutamate residues on the enzymatic properties of the activated blood clotting factor X

342

Biochimica et Biophysica Acta, 533 (1978) 342--354 © Elsevier/North-Holland Biomedical Press

BBA 37901 THE E F F E C T OF 7-CARBOXYGLUTAMATE RESIDUES ON THE ENZYMATIC PROPERTIES OF THE ACTIVATED BLOOD CLOTTING FACTOR X I. ACTIVITY TOWARDS SYNTHETIC SUBSTRATES

M.J. LINDHOUT, B.H.M. KOP-KLAASSENand H.C. HEMKER Department of Biochemistry, Biomedical Centre, State University Limburg, Maastricht (The Netherlands)

(Received November 2nd, 1977)

Summary The esterolytic and amidolytic properties of activated blood coagulation factor X (factor Xa) and the analogous decarboxy species were compared in order to find out if the 7-carboxyglutamic acid residues influence the function of the active centre. It was found that the two proteins (1) showed similar kinetic parameters when titrated with p-nitrophenyl-p'-guanidinobenzoate hydrochloride (2) had a similar Km and kcat for various synthetic chromogenic tri- and tetrapeptides and (3) were inhibited in the same way by benzamidine. Further it was observed that (4) Ca z÷ inactivates factor Xa, but has no influence on the amidase activity of decarboxyfactor Xa (5) factor V prevents Ca:+-induced inactivation of factor Xa but does not influence the amidase activity of both factor Xa and decarboxyfactor Xa. We conclude that the interaction of the 7-carboxyglutamic acid residues with Ca 2÷ in factor X has no measurable influence on the properties of the active site per se.

Introduction

Factor X~ is a protease belonging to the chymotrypsin family. With thrombin, plasmin, trypsin and chymotrypsin it shares many structural and functional features. It can be safely assumed that a similar catalytic mechanism, involving the "charge relay s y s t e m " Asp-His-Ser and c o m m o n features in the three dimensional structure are shared by these mammalian serine proteases [1--4]. Amino acid sequences in the region surrounding the reactive serines in the heavy chain of factor Xa are f o u n d to be homologous with the same regions of trypsin, chymotrypsin-A, elastase and thrombin [4--9]. Like trypsin, acti-

343 vated factor X is inhibited by soybean trypsin inhibitor [10,11] diisopropylphosphofluoridate [11,12] and synthetic aromatic amidine and guanidino inhibitors [ 13 ]. Factor Xa catalyzes the hydrolysis of various synthetic substrates such as N-a-benzoyl-L-arginine ethylester [14], N-a-p-tolyene sulfonyl-L-arginine methylester [11,15], p-nitrophenyl-p'-guanidinobenzoate [16] and benzoxyisoleucyl-glutamyl-glycyl-arginyl-p-nitroanilide [17]. Contrary to other known serine proteases (with the exception of clotting factor IXa) factor Xa has unique structural feature in that it possesses several couples of ~/-carboxyglutamic acid residues in the N-terminal part of its smaller chain. Activated factor X also can be considered a protease with trypsin-like specificity in that it selectively attacks amido bonds adjacent to arginine. Unlike trypsin, the Nacylamino acid esters containing lysine are less readily hydrolized by factor Xa than the arginine-containing esters [ 14 ]. When acting on its natural substrate (prothrombin) factor Xa cleaves only 274 275 323 324 two peptide bonds adjacent to arginine, the Arg-Thr and Arg-Ile bonds [18-21], whereas there are 75 bonds which in principle, must be considered susceptible. The highly restricted substrate specificity of factor X~ is an interesting, but hardly investigated, feature. Experiments comparing factor Xa to trypsin with respect to their inhibition by various amidines and guanidines, carried out by Johnson et al. [13] suggest that factor Xa contains a primary substrate binding site equal to that of trypsin in potential binding energy and a secondary substrate binding site responsible for the specificity of factor X~. It can be safely assumed that apart from the active site-vulnerable site interaction in a protease and its protein substrate "subsite" interaction are largely responsible for highly specific interactions like these of factor X~ and prothrombin [22--25]. It is the purpose of the experiments described in this paper to elucidate a possible role of these ")'-carboxyglutamate residues in the catalytic activity of factor Xa by comparing it with the activity of decarboxyfactor X~. This protein, isolated from dicoumarol-treated cattle is completely identical to factor X~ but for the presence of glutamyl instead of "y-carboxyglutamyl residues. Materials and Methods Materials and methods, except for those described below, are reported earlier [26]. p-Nitrophenyl-p'-guanidinobenzoate hydrochloride (NPGB) was purchased from Biochemical Nutrition Corp. Benzoxy-phenylalanyl-valyl-arginyl-p-nitroanilide (Bz-ehe-Val-Arg-pNA) and benzoxy-isoleucyl-glutamyl-glycyl-arginyl-pnitroanilide (Bz-Ile-Glu-Gly-Arg-pNA) were products of A.B. Bofors, Nobel Pharma, Sweden. Activated factor X and activated decarboxyfactor X were prepared with the method descrived in a preceding paper [26]. Factor V was prepared according to Smith and Hanahan [41]. A specific activity of 180 U/mg was obtained after activation with a trace of factor V activator from Russell's Viper venom. No other procoagulant activities could be found in this preparation.

344

Titration o f factor Xa and decarboxyfactor Xa Titration o f activated factor X and activated d e c a r b o x y f a c t o r X was perf o rmed according to Smith [16]. In a typical titration the sample cuvette contains~150 pg o f f act or Xa or 180 pg of d e c a r b o x y f a c t o r Xa in 300 pl of 0.1 M sodium barbital buffer, pH 8.3. The reference cuvette contains the same volume o f buffer solution alone. To each cuvette 3 ~l of a 0.01 M solution of NPGB in dimethyl-formamide-acetonitrile ( 1 : 3 , v/v) were simultaneously added. The reaction was followed at 410 nm in an Aminco DW-2 spectrophot o m e t e r at 25°C. The c o n c e n t r a t i o n of p- ni trophenol released during the reaction was calculated from the zero time intercept of the extrapolated steady state line. In the Aminco DW-2 s p e c t r o p h o t o m e t e r , e4~0 for p-nitrophenol at pH 8.3 was calculated to be 17 500. Determination o f kinetic constants K S and k2 The presteady-state reaction constants of factor X a and d e c a r b o x y f a c t o r Xa with NPGB were det er m i ned according to Bender et al. [27]. E n z y m e concentrations were 3.2 • 10 -6 M factor Xa and 2.2 • 10 -6 M d e c a r b o x y f a c t o r Xa. The NPGB concentrations ranged from 3 . 3 . 10-SM to 1 3 . 3 . 10 -s M. The operational first order rate constant for the presteady-state reaction (b) was determined at each c o n c e n t r a t i o n of NPGB by linear regression analysis. The rate constant o f acylation (k2) and the association constant Ks of the e n z y m e • substrate co mp lex were determined f r om the intercept and slope of a plot of lib versus 1 / [ N P G B ] . Determination o f k3 and Kpb Acylation of f a c t or X a and d e c a r b o x y f a c t o r Xa was p e r f o r m e d by addition o f 10 pl o f 0.01 M NPGB to 1.2 ml of a solution of factor Xa or decarboxyfactor X~ in 0.1 M sodium barbital buffer, pH 8.3. The final c o n c e n t r a t i o n of NPGB was 8.3. 10 -5 M. The final e n z y m e concentrations were 3.2 • 10 -6 M factor Xa and 2.2 • 10 -6 M d e c a r b o x y f a c t o r Xa. The a c y l e n z y m e solutions with excess NPGB were applied to a col um n (0.9 × 30 cm) of Sephadex G-25 equilibrated with 0.1 sodium barbital buffer, pH 8.3. Enzyme-containing fractions were collected and incubated at 25°C. At several intervals aliquots were removed from the incubation m i xt ur e and assayed for amidase activity with Bz-Ile-Glu-Gly-Arg-pNA. The time by which the a c y l e n z y m e had sunk into the column was taken as zero time. The deacylation rate constant (k3) was determined from the slope of In [Eo]/([Eo] -- [Et]) versus time, where [E0] is the c o n c e n t r a t i o n o f a c y l e n z y m e at zero time and [Et] the c o n c e n t r a t i o n of deacylated e n z y m e at time t. Amidase activity is det erm i ned as described in this section. The rate cons t a nt for the postburst p-nitrophenol p r o d u c t i o n (Kpb) is determined at 1.6 • 10 -4 M NPGB. Amidase activity assay o f factor Xa and decarboxyfactor X a The initial rates o f hydrolysis o f the amides N-benzoyl-L-phenylalanyl-Lvalyl-L-arginine-paranitroanilide-HC1 and N-benzoyl-L-isoleucyl-L-glutamylglycyl-L-arginine-paranitroanilide-HC1 by factor Xa and d e c a r b o x y f a c t o r Xa were measured in Tris-imidazole buffer, pH 8.2, and ionic strength o f 0.15 at 37°C by determining the increase of p-nitroaniline absorbance in the Aminco

345 DW-2 spectrophotometer operating in the dual wavelength mode with hr = 3 4 4 n m and Xs = 391 nm at 37°C. The e391.344 of p-nitroaniline in the buffer solution of pH 8.2 was determined as 11 400 A/mol. Results

Titration o f factor Xa and decarboxyfactor Xa Functional enzyme concentration. Factor Xa and decarboxyfactor Xa were titrated with NPGB as described under Materials and Methods. As shown in Fig. 1 the presteady-state p-nitrophenol production was complete after about 2 min for both factor Xa and decarboxyfactor Xa and was followed by a very low linear postburst production of p-nitrophenol. From this type of curve the presteady-state production of p-nitrophenol (~) was determined [ 16]. As can be seen from Table I, ~ does not vary with the NPGB concentration. This fact, and the low postburst production of p-nitrophenol indicate that So > > KM and k2 > > k3. Therefore [E0] ~ ~ and the molarity of the enzyme solutions can be calculated as (3.1 + 0.1) • 10 -6 M and (2.1 -+ 0.1) • 10 -6 M for factor X a and decarboxyfactor Xa, respectively. Determination o f kinetic constants. The kinetic constants of the hydrolysis of NPGB catalyzed by factor Xa and decarboxyfactor Xa are summarized in Table II. The linear relationship between 1/b versus 1/NPGB (Fig. 2) and ln[Eo/ES'] versus time (Fig. 3) show that the kinetics of both factor Xa and decarboxyfactor Xa reacting with NPGB are adequately described by the theory of Bender et al. [ 27]. The rate constant for the postburst p-nitrophenol production, Kpb, determined at an NPGB concentration of 1 . 6 . 1 0 - 4 M was about twice the deacylation constant k3 for both factor X a and decarboxyfac-

6.0-

5o

S J°-- ~ - ~ - ~ ~

40

f'~--~----~J

% '~ 30 o uJ •::z 2.0

1.0-

0.'5

110

115

210 215 time (rain)

3.0

3.5

4'.0

F i g . 1. T i m e c o u r s e s o f t h e r e a c t i o n b e t w e e n p ' - n i t r o p h e n y l - p - g u a n i d i n o b e n z o a t e (NPGB) and factor Xa (open circles) and decarboxyfactor X a ( s o l i d c i r c l e s ) . P r o c e d u r e is as d e s c r i b e d u n d e r M a t e r i a l s a n d M e t h o d s . T h e a c t u a l p r o t e i n c o n c e n t r a t i o n is 3 . 6 • 1 0 - 6 a n d 3,1 • 1 0 - 6 M f o r f a c t o r X a a n d d e c a r b o x y f a c t o r __Xa, r e s p e c t i v e l y . T h e N P G B c o n c e n t r a t i o n is 9 . 9 • 1 0 - s M.

346 TABLE

l

TITRATION

OF

FACTOR

X a AND DECARBOXYFACTOR

X aWITH

p-NPGB:

INDEPENDENCE

OF

AND p-NPGB CONCENTRATION F i n a l p-NPGB c o n c e n t r a t i o n

Presteady-state

in c u v e t t e (M)

p-nitrophenol

Functional

production

Factor X a

enzyme

concentration

Ur) AI'; 4 t 0 Dccarboxyfactor X a

(~zM)

Factor X a

Decarboxyfactor X a

3.3

1 0 -5

5.30 " 10 2

3.64 " 10 -2

3.03

2.08

6.6

10 -5

5.68 " 10 .2

4 . 0 6 " 10 -2

3.25

2.32

9.9

1 0 -5

5 . 5 3 " 10 -2

3.85"

10 -2

3.16

2.20

13.2

10 -s

5 . 2 8 " 10 -2

3.94"

10 -2

3.02

2.25

TABLE II KINETIC CONSTANTS OF REACTION OF FACTOR Xa AND DECARBOXYFACTOR Xa WITH p-NPGB Constant

Factor X a

Decarboxyfactor

Ks

4.2'

4.0-

I~ 2 /"3

0.19 s i 4 . 2 " 1 0 - 5 - s -1

0 . 1 5 s -1 3.0- 10 -5.

k2/;¢ 3 KM Kpb

4.5" 10 -3 9.3 • 10 -8 M

5.0' 8.0"

10 -3 10 -8 M

8.0'

7.0'

10 -5 M

10 -4 M

10 -5 M

Xa

10 -4 M s -1

60-

0.8

0.6

o~

uJ~° 0.4 c

1°/~ti 0.5

. . . . . 1.0

1.5 1

2.0

(M-1)x10-4

0.2

2.5

3.0

100

200 incubation

300

400

time (min)

F i g . 2. P l o t o f t h e f i r s t o r d e r r a t e c o n s t a n t o f t h e p r e s t e a d y s t a t e r e a c t i o n o f f a c t o r X a ( o p e n c i r c l e s ) a n d decarboxyfactor X a (solid circles) versus the reciprocal p-NPGB concentration. Fig. 3. D e a c y l a t i o n of g u a n i d i n o b e n z o y l - f a c t o r X a (open circles) and guanidinobenzoyl-decarboxyfactor X a (solid circles). Experimental details are described under Materials and Methods.

347 tor Xa. However, the ratio k2/k 3 is very large; therefore the postburst p-nitrophenol production had no serious effect on the accuracy of the factor Xa and decarboxyfactor Xa titration under the conditions used.

Lt3

8.o-I

L,~

A

8.0-

x

E

~. 6.0-

"5 F

, 4.0

~

f o 4.02.0-

S

0

4

8 1__ ( M-1 )

12

16

0

i

,

,

i

4

8

12

16

1__ (M-l) x 10-3 So

x10-3

So

D

/ 200-

200"

16.0"

16.0-

12,0-

120-

×

E •-"

-I~'° 80-

i:.°

4.0

f

0

80.

S 1.0

2.0

30

I (M-1)x 10-4 So

40

i

1.0

,

2.0

,

3.0

r

4.0

1__ (M-1)xlO-4 So

F i g . 4. L i n e w e a v e r - B u r k p l o t s f o r f a c t o r X a a n d d e c a r b o x y f a c t o r X a using Bz-Phe-Val-Arg-p-NA and B z - I l e - G l u - G l y - A r g - P N A . A . A m i d o l y s i s o f B z - I l e - G l u - G l y - A r g - p N A b y d e c a r b o x y f a c t o r X a. E n z y m e c o n c e n t r a t i o n , 5.0 • 1 0 - 9 M ( o p e n c i r c l e s ) a n d 2 . 5 • 1 0 - 9 M ( s o l i d c i r c l e s ) . B. A m i d o l y s i s o f B z - I l e - G l u - G l y A r g - p N A b y f a c t o r X a. E n z y m e c o n c e n t r a t i o n , 4 . 0 • 1 0 - 9 M ( o p e n c i r c l e s ) a n d 2 . 0 • 1 0 - 9 M ( s o l i d c i r c l e s ) . C. A m i d o l y s i s o f B z - P h e - V a l - A r g - p N A b y d e c a r b o x y f a c t o r X a. E n z y m e c o n c e n t r a t i o n s , 9 . 0 . 1 0 -8 M ( o p e n c i r c l e s ) a n d 6.1 • 1 0 - 8 M ( s o l i d c i r c l e s ) , D . A m i d o l y s i s o f B z - P h e - V a l - A r g - P N A b y f a c t o r X a. E n z y m e c o n c e n t r a t i o n s , 1.9 • 10 -7 M ( o p e n circles) a n d 1.3 • 10 -7 M (solid circles). E x p e r i m e n t a l c o n d i t i o n s as d e s c r i b e d u n d e r M a t e r i a l s a n d M e t h o d s .

3~

TABLE

III

KINETIC

CONSTANTS

FACTOR

X AND ACTIVATED

F'OR 'rife

Substrate

Factor

Bz-Phe-Val-Arg-pNA Bz-Ile-Glu-Gly-Arg-pNA

Results of duplicate

HYDROLYSIS

OF SYNTHETIC

DECARBOXYFACTOR

AMIDE

SUBSTRATES

BY ACTIVATFA)

X ~

Xa

Dcearboxyfaetor

Xa

K M (M)

hca t ( r a i n 1)

K M (M)

h c a t ( r a i n 1)

(0.9 - 0.1) . 10 5 ( 0 . 4 , t : 0 . 0 6 ) • 10 -3

16 - 1 ( 6 . 6 _* 0 . 1 ) . 1 0 3

(I.2 " 0.1) • 1 0 -5 ( 0 . 5 0 ~ 0 . 0 6 ) • 1 0 ,3

55 ' 5 (5.7 t 0.1) • 103

experiments

using two different enzyme concentrations.

Amidase activity of factor X~ and decarboxyfactor X~ Determination of the kinetic constants. The Lineweaver-Burk plots [28] for factor X~ and d e c a r b o x y f a c t o r Xa with Bz-Phe-Val-Arg-pNA and Bz-Ile-GluGly-Arg-pNA are shown in Fig. 4, A--D. The kinetic constants are summarized in Table III and were determined from slope and intercept by regression analysis of each of data. The functional e n z y m e concentrations determined by NPGB titration, instead of the e n z y m e concent rat i on based on protein concentration were used for calculation of kcat = V/[Eo]. An excess, at least 150-fold for Bz-Phe-Val-Arg-pNA and at least 10 000-fold of Bz-Ile-Glu-Gly-Arg-pNA over e n z y m e c o n c e n t r a t i o n was used. pH dependence. For both factor X, and d e c a r b o x y f a c t o r X~ the pH optimum was f o u n d to be between 8.0 and 8.5 (Fig. 5). It also appears from these

5.0"

4.0O x 3.0-

o

~91"'2.O_

1.0-

-4/ . 5.0

.

. 6.0

. 7.0

. 8.0

. 9.0

10.0

pH Fig. 5. p i t o p t i m a of f a c t o r X a (~) a n d d e c a r b o x y f a c t o r X a (0) with Bz-ne-Glu-Gly-Arg-pNA. The pit d e p e n d e n c e w a s d e t e r m i n e d in T r i s - i m i d a z o l e h u f f e r e s a t v a r y i n g p H , i o n i c s t r e n g t h o f 0 . 1 5 a t 3 7 ° C . S u b strate concentration was 1.2 • 10 -4M, enzyme concentrations were: factor Xa, 1.2 • 10 -8 M and decarb o x y f a e t o r X a , 1.1 • 1 0 - 8 M. A m i d a s e a c t i v i t y a s s a y as d e s c r i b e d u n d e r M a t e r i a l s a n d M e t h o d s .

349 B

A

6.0io

~ ,

,-T

o

o

$

5.5-

10-{

I

5.0.

8-1 × 4.5' 6-t

.E E

-° 4.0o

o

4-

%

3.5-

2-

~e

3.0,

20

40

incubation

60

time

80

(min)

100

0.5

1.0

1.5

2.0

1 (M-l) xlO 4 S

F i g . 6. E f f e c t o f C a 2+ o n t h e a m i d a s e a c t i v i t y o f f a c t o r X a a n d d e c a r b o x y f a c t o r X a with Bz-Ile-Glu-GlyArg-pNA. A. Time courses of inactivation of factor Xa, 50 pg/ml (solid circles) and decarboxyfactor Xa, 6 5 p g / m l ( s o l i d t r i a n g l e s ) b y Ca 2+ ( 5 m M ) . T h e s a m e e x p e r i m e n t s i n t h e a b s e n c e o f Ca 2+ a r e p r e s e n t e d b y open circles for factor X a and open triangles for decaxboxyfactor X a. A t i n t e r v a l s a l i q u o t s ( 4 #1) w e r e removed from the incubation mixture containing enzymes in a buffer of 0.025 M Tris-imidazole/O.1 M NaC1, pH 8.2, Experiments were performed at 37°C. Amidase activity was measured as described under M a t e r i a l s a n d M e t h o d s . B. D o u b l e r e c i p r o c a l p l o t s o f t h e a m i d o l y s i s o f B z - I l e - G l u - G l y - A r g - p N A b y f a c t o r X a. F a c t o r X a w a s p r e i n c u b a t e d i n t h e p r e s e n c e o f C a 2+ f o r 6 0 m i n ( s o l i d c i r c l e s ) a n d a b s e n c e o f C a 2+ (open circles). Experimental conditions as described under Fig. 6A.

results t h a t v a r i a t i o n o f t h e p H in t h e r a n g e i n d i c a t e d h a d n o i n f l u e n c e on t h e kca t ( f a c t o r X a / k c a t ( d e c a r b o x y f a c t o r Xa) ratio. E x p e r i m e n t a l c o n d i t i o n s w e r e as d e s c r i b e d u n d e r Fig. 5. E f f e c t o f Ca 2÷ on amidase activity. As s h o w n in Fig. 6A, t h e initial v e l o c i t y o f t h e h y d r o l y s i s o f B z - I l e - G l u - G l y - A r g - p N A c a t a l y z e d b y f a c t o r Xa, decreases d u r i n g i n c u b a t i o n in t h e p r e s e n c e o f Ca 2÷ (5 m M ) . A f t e r a b o u t 60 m i n t h e a m i d a s e activity r e m a i n s u n a l t e r e d . T h e a m i d a s e activity t h e n f o u n d is o n e h a l f o f t h e c o n t r o l value. Double reciprocal plots of the hydrolysis of Bz-Ile-Glu-Gly-Arg-pNA by f a c t o r Xa, p r e i n c u b a t e d d u r i n g 90 m i n in t h e p r e s e n c e o f 5 m M CaC12 a n d fact o r X a t r e a t e d in t h e s a m e w a y in t h e a b s e n c e o f Ca 2÷ are s h o w n in Fig. 6B. It was f o u n d t h a t in t h e a b s e n c e o f Ca 2÷ t h e V v a l u e was 2.5 • 10 -s m o l • m i n -t and in t h e p r e s e n c e o f Ca 2÷ was 1.2 • 10 -s m o l • m i n -l . T h e g M value f o r fact o r Xa p r e i n c u b a t e d with Ca 2÷ was t h e s a m e as t h a t o f f a c t o r Xa p r e i n c u b a t e d in t h e a b s e n c e o f Ca 2÷. U p o n i n c u b a t i o n o f d e c a r b o x y f a c t o r Xa with Ca 2÷ u n d e r identical c o n d i t i o n s as d e s c r i b e d f o r f a c t o r Xa n o a l t e r a t i o n was f o u n d in t h e a m i d a s e activity. E f f e c t o f f a c t o r V a on amidase activity. F a c t o r Va has n o significant e f f e c t u p o n t h e K M a n d V values of t h e h y d r o l y s i s o f B z - I l e - G l u - G l y - A r g - p N A c a t a l y z e d b y f a c t o r Xa a n d d e c a r b o x y f a c t o r Xa u n d e r t h e c o n d i t i o n s d e s c r i b e d in t h e legend to Fig. 7. H o w e v e r , n o decrease in a m i d a s e a c t i v i t y o f f a c t o r Xa

;350

18-

/

16-

14"

o x

A

=

12"

i ~

10-

8-

/

•

6"

4"

1(M-1)xlO-3 S

F i g . 7. E f f e c t o f f a c t o r V a o n t h e a m i d a s e a c t i v i t y o f f a c t o r X a a n d d e e a r b o x y f a c t o r X a using Bz-lle-GluG l y - A r g - p N A as s u b s t r a t e . L i n e w e a v e r - B u r k p l o t s w e r e c o n s t r u c t e d f r o m d a t a o b t a i n e d b y a m i d o l y s i s o f B z - l l e - G l u - G l y - A r g - p N A b y 1, f a c t o r X a ( ~ ) ; 2, f a c t o r X a p r e i n c u b a t e d f o r 6 0 m i n in t h e p r e s e n c e o f Ca 2+ ( 5 r a M ) , ( . ) ; 3, f a c t o r X a i n t h e p r e s e n c e o f f a c t o r V a ( 1 0 U / m l (,~); 4, f a c t o r X a in t h e p r e s e n c e o f Ca 2+ (5 r a M ) a n d f a c t o r V a ( 1 0 U / m l ) ( i ) ; 5, d e c a r b o x y f a c t o r Xa, (~); 6, decaxboxyfactor X a in the presence of factor V a (10 U/ml), (i). Enzyme concentrations w e r e : f a c t o r X a , 1.8 • 1 0 - 8 M a n d d e c a r boxyfactor X a , 1 . 0 • 1 0 - 8 M. S u b s t r a t e c o n c e n t r a t i o n s ranging from 8.7 .10 -5 to 2.3 . 10 -4M. Experim e n t a l c o n d i t i o n s w e r e t h e s a m e as d e s c r i b e d u n d e r F i g . 6.

u p o n incubation with Ca ~+ (5 mM) was f o u n d when factor V a (1.0 mg/ml) was present in the incubation mixture. This effect could however, be duplicated by the addition of bovine serum albumin to a concent rat i on o f 0.5 mg/ml (results n o t shown). Inhibition o f amidase activity by benzamidine. A plot of the reciprocal initial rates of factor Xa and d e c a r b o x y f a c t o r Xa hydrolysis of Bz-Ile-Glu-GlyArg-pNA {l/v0) versus inhibitor c o n c e n t r a t i o n (I) at different substrate concentrations [29] is shown in Fig. 8. For both fact or Xa and d e c a r b o x y f a c t o r Xa straight lines were obtained. The inhibition by benzamidine was observed as competitive. Competitive inhibition was also f o u n d in a double reciprocal plot o f v0 versus substrate c o n c e n t r a t i o n at different inhibitor concentrations. The Ki values for inhibition of the hydrolysis of Bz-Ile-Glu-Gly-Arg-pNA determined from Fig. 8 by regression analysis were f o u n d to be 2 . 4 - 1 0 -4 and 3 . 0 , 10 -4 M for factor X a and d e c a r b o x y f a c t o r Xa, respectively.

351

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I(M)xlO4 Fig. 8. I n h i b i t i o n o f a m i d a s e a c t i v i t y o f f a c t o r X a a n d d e c a r b o x y f a c t o r X a b y b e n z a m i d i n e . D i x o n p l o t was c o n s t r u c t e d by plotting the reciprocal initial rate of Bz-Ile-Glu-Gly-Arg-pNA hydrolysis by factor X a ( A ) a n d d e c a r b o x y f a c t o r X a (B) vel'sus i n h i b i t o r c o n c e n t r a t i o n at f i x e d c o n c e n t r a t i o n s o f s u b s t r a t e as i n d i c a t e d . E n z y m e c o n c e n t r a t i o n s w e r e 1.0 • 10 -8 M f o r d e c a r b o x y f a c t o r X a a n d 0 . 4 • 10 -8 M f o r f a c t o r X a. A m i d a s e a c t i v i t y w a s m e a s u r e d as d e s c r i b e d u n d e r M a t e r i a l s a n d M e t h o d s .

Discussion Titani [30] has shown that there exists a high degree of identity (greater than 55%) between the heavy chain of factor X a and trypsin in a region (73 residues) which includes the active site serine. All components of the "charge-

352 relay system" of pancreatic serine proteases are present in the corresponding loci of the heavy chain of factor Xa. However, the trypsin-like proteases of the blood coagulation system, e.g. thrombin, factor IXa and factor Xa, possess a narrow substrate specificity compared to trypsin. It is also known that these proteases possess an extra chain not found in trypsin and it is suggested that this chain might be the source of the specificity [8,31]. Recently, Hageman [32] found evidence that the A-chain of thrombin does not play a significant role in determining the catalytic specificity of thrombin. In fact, by studying the specificity of thrombin and factor Xa towards small synthetic substrates it was found that there exists a narrow tolerance of thrombin and factor X a for recognition of amino acids in either of the two adjacent aminoterminal residues of the Arg-X peptide bond to be split. For thrombin Val or Ala and for factor X a an acidic amino acid (Glu) is found in either of the two residues [18,33,34]. It was our purpose to elucidate the role of 7-carboxyglutamate residues in the catalytic action of factor Xa by comparing the catalytic properties of the native molecule (factor Xa) and an analogous decarboxy species. This is immediately linked to the question of the role of Ca z÷, since the binding of these ions to the 7-carboxyglutamate residues is probably essential in their function. There is no d o u b t that Ca 2÷ acts in blood coagulation, because it binds factor Xa to a phospholipid-water interface [35]. But it is possible that it has more than one function in the catalytic reactions of factor Xa. This is the more probable as the binding of Ca 2÷ to factor X brings about conformation changes [36]. The hydrolysis of NPGB, catalyzed by factor X, and deearboxyfactor Xa revealed identical titration curves (Fig. 1). Therefore, roughly the same kinetic parameters, e.g. association constant (Ks) of the enzyme • substrate complex formation, acylation rate constant (k2) and deaeylation rate constant (k3) were found (Table II). We concluded from these results that all functional species of the active site of deearboxyfaetor Xa were intact. All kinetic parameters were found in a good agreement with those reported for factor Xa by Smith [16]. Our investigations on the catalytic efficiency of factor X a and decarboxyfaetor X~ were extended with two more substrates: Bz-Phe-Val-Arg-pNA and Bz-Ile-Glu-Gly-Arg-pNA. Using a steady-state kinetic method, kinetic parameters of the activity of factor Xa and deearboxyfactor Xa towards Bz-Phe-Val-Arg-pNA and Bz-IleGlu-Gly-Arg-pNA were determined (Table III). Our results suggest that both enzymes have an identical affinity towards Bz-Phe-Val-Arg-pNA as measured by KM. The KM values f o u n d were of the same order of magnitude as was reported for trypsin: 6.7 • 10 -s M [34] and thrombin 4.8 • 10 -s M [25]. Also, the affinity of factor Xa and deearboxyfaetor X a for Bz-Ile-Glu-Gly-Arg-pNA were found to be equal, (KM values 4.4 • 10 -4 and 5.0 • 1 0 - 4 M respectively) and to agree very well with the data supplied by Kabi Diagnostica, Sweden (3.0" 10 -4 M). A comparison of the K M values determined for both substrates shows that the enzymes bind Bz-Phe-Val-Arg-pNA more effectively than Bz-Ile-Glu-GlyArg-pNA by one order of magnitude. However, under the conditions used in these experiments factor X~ hydrolyzes Bz-Ile-Glu-Gly-Arg-pNA much faster (400 times) than it does Bz-Phe-Val-Arg-pNA and so does decarboxyfactor X~ (100 times faster).

353 This discrepancy found between factor Xa and decarboxyfactor Xa was to the less catalytic efficiency of factor Xa towards Bz-Phe-Val-Arg-pNA in comparison to decarboxyfactor Xa (kcat values 16 m i n - ' and 55 min-'respectively). This was not due to the presence of thrombin in the decarboxyfactor Xa preparation. Also the difference could not be due to the presence of contaminant proteins. The ratio protein concentration and functional enzyme concentration is 95/100 and 75/100 for the normal factor and the decarboxyfactor, respectively. This would induce approx. 20% difference in the kc~t at the utmost. Recently, Suomela [37] reported that factor Xa had a considerable susceptibility towards the substrate Bz-Ile-Glu-Gly-Arg-pNA. Among the other coagulation factors only thrombin appeared to hydrolyze this substrate at a low rate. A comparison of the inhibition constants of factor Xa and decarboxyfactor Xa for benzamidine, using Bz-Ile-Glu-Gly-Arg-pNA as the substrate, shows that both enzymes have an identical affinity for the inhibitor and that inhibition was competitive (Fig. 8). Ca 2+ was found to be a non-competitive inhibitor of the amidase activity of factor Xa towards Bz-Ile-Glu-Gly-Arg-pNA. The time course of inactivation suggests that it finally reaches an equilibrium. It was already shown by Jesty [38] that Ca 2+ mediates a concentration-dependent association of factor Xa, resulting in the precipitation of factor X~ aggregates. Since it has been shown that Ca 2+ has no effect upon the amidase activity of decarboxyfactor X~, the results suggest that the 7-carboxyglutamate residues are involved in the Ca2+-mediated association of factor Xa. Of particular interest seemed the observation that factor Va protects the Ca2+-induced inactivation of factor Xa {Fig. 7). However, the finding that bovine serum albumin also protects factor Xa from inactivation suggests that a nonspecific protein-protein interaction prevents inhibition of factor X~. In the absence of Ca 2+, factor V~ does not enhance the amidase activity. The increase in the catalytic efficiency of factor Xa towards p-toluene sulfonyl-Larginine methyl ester caused by factor V as reported by Colman [39] may therefore be due to the presence of Ca 2+ in his assay mixture. We conclude from these results that the 7-carboxyglutamate residues have no significant influence on the catalytic efficiency of factor X a towards small synthetic substrates. Moreover, Ca 2+ has no effect on the active centre per se. Investigations on the activation of prothrombin by factor Xa and decarboxyfactor X a clearly demonstrate that, unlike the case of decarboxyfactor X~, Ca 2+ enhances the activation of prothrombin by factor X a. This suggests a role of the 7-carboxyglutamate residues in subsite action in addition to the well documented role in the Ca2+-mediated binding of factor Xa to phospholipids (Lindhout, M.J., Kop-Klaassen, B.H.M. and Hemker, H.C. {1978), in preparation). References 1 Blow, D.M., Birktoff, J.J. and Hartley, B.S. (1963) Nature 2 2 1 , 3 3 7 - - 3 4 0 2 Freer, S.T., Krant, J., Robertus, J.D., Wright, H.T. and Xuong, N.H. (1970) Biochemistry, 1997-2009 3 S h o t t o n , D.M. and Hartley, B.S. (1970) Nature 225, 802--806 4 Titani, K., Hermodson, M.A., Fujikawa, K., Ericsson, L.H., Walsh, K.A., Neurath, H. and Davie, E.W. (1972) Biochemistry 11, 4 8 9 9 - - 4 9 0 3

354

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

28 29 30 31 32 33 34 35 36 37 38 39 40 41

Walsh, K.A. and Neurath, H. (1964) Proc. Natl. Acad. Sci. U.S.A. 52, 884--889 Hartley, B.S. (1970) Phil. Trans. R. Soc. Lond. B. 257, 77--87 Hartley, B.S. and Shotton, D.M. (1971) Enzymes 3, 323--373 Magnusson, S. (1971) Enzymes 3 , 2 7 7 - - 3 2 1 De Haen, C., Neurath, H. and Teller, D.C. (1975) J. Mol. Biol. 92, 225--259 Lundblad, R.L. and Davie, E.W. (1965) Biochemistry 4, 113--120 Jack so n, C.M. and Hanahan, D.J. (1968) Biochemistry 7, 4 5 0 6 - - 4 5 1 7 Fujikawa, K., Legaz, M.E. and Davie, E.W. (1972) Biochemistry 11, 4 8 9 2 - - 4 8 9 9 Joh nso n, V.A. and Smith, R.L. (1976) Arch. Biochem. Biophys. 175, 190--195 Adams, R.W. and Elmore, D.T. (1971) Biochem. J. 124, 66p Esnouf, M.P. and Williams, W.J. (1962) Biochem. J. 84, 62--71 Smith, R.L. (1973) J. Biol. Chem. 248, 2418--2423 Kosow, D.P. (1976) Thrombos. Res. 9, 565--573 Magnusson, S., Sottrup-Jensen, L., Petersen, T.E. and Claeys, H. (1975) in P r o t h r o m b i n and Related Coagulation Factors, Hemker, H.C. and V e l t k a m p , J.J., eds.), pp. 2 5 - 4 6 , Leiden University Press Owen, W.J., Esmon, C.T. and Jackson, C.M. (1974) J. Biol. Chem. 249, 594---605 Kisiel, W. and Hanahan, D.J. (1974) Biochem. Biophys. Res. Commun. 59, 570--577 Esmon, C.T., Owen, W.G. and Jackson, C.M. (1974) J. Biol. Chem. 249, 6 0 6 - 6 1 1 Hageman, T.C. and Scheraga, H.A. (1974) Arch. Biochem. Biophys. 164, 707--715 Berger, A. and Schleehter, I. (1970) Phil. Trans. R. Soe. Lond. Ser. B 2 5 7 , 2 4 9 Thomp so n, A.R. (1976) Biochim. Biophys. Acta 422, 200--209 Gorrnan, J.J. (1975) Bioehim. Biophys. Acta 4 1 3 , 2 7 3 - - 2 8 2 L i n d h o u t , M.J., KoP-Klaassen, B.H.M. and Hemker, H.C. (1978) Biochim. Biophys. Acta 5 3 3 , 3 2 7 - 341 Bender, M.L., Begu~-Canton, M.L., Blakely, R.L., Brubacher, L.J., Feder, J., Gunter, C.R., Kezdy, F.J., KoUheffer, J.V., Marchall, T.H., Miller, C.G., Roeske, R.W. and Stoops, J.K. (1966) J. Am. Chem. Soc. 88, 5890--5913 Lineweaver, H. and Burk, D. (1934) J. Am. Chem. Soc. 5 6 , 6 5 8 - - 6 6 6 Dixon, M. (1953) Biochem. J. 55, 170--171 Titani, K., Fujikawa, K., Enfield, D.L., LoweLl, H.E., Waish, K.A. and Neurath, H. (1975) Proc. Natl. Acad. Sci. U.S. 72, 3082--3086 Fujikawa, K., Legaz, M.E., Kato, H. and Davie, E.W. (1974) Biochemistry 13, 4 5 0 8 - - 4 5 1 6 Hageman, T.C., Endxes, G.F. and Scheraga, H.A. (1975) Arch. Bioch. Biophys. 1 7 1 , 3 2 7 - - 3 3 6 Takagi, T. and Doolittle, R.F. (1974) Biochemistry 1 3 , 7 5 0 - - 7 5 6 Svendsen, L., Blomb~/ck, B., Blomb~/ck, M. and Oisson, P.I. (1972) Thrombos. Res. 1 , 2 6 7 - - 2 7 8 Gitel, S.N., Owen, W.G., Esmon, C.T. and Jackson, C.M. (1973) Proc. Natl. Acad. Sci. U.S. 70, 1344-1348 Lindhout, M.J. and Hemker, H.C. (1978) Biochim. Biophys. Acta 533, 318--326 Suomela, H., Blomb~ick, M. and Blomb~ick, B. (1977) Thrombos. Res. 10, 267--281 Jesty, J. and Esnouf, M.P. (1973) Biochem. J. 1 3 1 , 7 9 1 - - 7 9 9 Colman, R.W. (1970) Brit. J. Haematol. 1 9 , 6 7 5 - 6 8 4 L i n d h o u t , M.J., Kop-Klaassen, B.H.M. and Hemker. H.C., in preparation Smith, C.M. and Hanahan, D.J. (1976) Biochemistry 15, 1830--1838