The action of thrombin on peptide p-Nitroanilide substrates

Btochtmtca et Btophysma Act~ 742 (1983) 539-557

539

Eisevaer B~omed~eal Press BBA31476

THE ACTION OF THROMBIN ON PEPTIDE p-NITROANILIDE SUBSTRATES SUBSTRATE SELECTIVITY AND EXAMINATION OF HYDROLYSIS UNDER DIFFERENT REACTION CONDITIONS RICHARD LOTTENBERG, JULIE A HALL, MOREY BLINDER, ELLEN P BINDER and CRAIG M JACKSON

Department of Btologtcal Chemistry, Dtwston of Biology and Btomedtcal Sciences, Washington Umverszty School of Medtcme, St Louts, MO 63110 ( U S A ) (Received May 17th, 1982) (Revtsed manuscript received November 9th, 1982)

Key words Thrombm, Poly(ethylene glycol), Substrate selectwtty, Pepttde p-nttroamhde, Hydrolysts

Kinetic parameters for the action of bovine ct-thrombin on 24 commercially available peptide p-nitroanilides have been determined. The selectivity constant, kcat/Km, ranges from 3.3.10 ! to 1.1. l0 s M - 1 . s - 1 for the poorest and the best substrates, respectively. The best substrates for thrombin were identified as those with arginine in the PI position, proline or a proline homolog in the P2 position, and an apolar amino acid in the P3 position. Quantitative distinction between lysine and arginine in the P! position and other amino acids in the Pz-P4 positions of the substrate is reported from the changes in the kinetic parameters lot substrates differing in only a single amino acid in these positions. Effects of NaCI, CaC!2 and poly(ethylene glycol) concentrations, pH and temperature on the action of thrombin on selected substrates have been assessed. A source of large systematic error in thrombin concentration estimates was identified as resulting from adsorption losses. These losses were eliminated by inclusion of poly(ethylene glycol) in dilution and reaction buffers.

Introduction

Structural studies of the serine protemases of the blood coagulation system [1,2] have demonstrated primary structure sirmlanties between them and trypsin and chymotrypsln. These slmllanUes are sufficiently great, parucularly within the actwe-slte regions and the regions that produce the fl-barrels of the pancreaUc serine proteinases, that three-dimensional models have been constructed for the coagulaUon proteases [3,4]. Development of peptlde p-nltroamlide substrates with poten-

Abbrevtauons Hepes, 4-(2-hydroxyethyl)-i-piperazaneethanesulfonic acid, NPGB, p-nltrophenyl-p-guamdlnobenzoate-HCl 0167-4838/83/0000-0000/$03 00 © 1983 Elsevier Blome&cal Press

Ually great selecuvity and sensitivity for thrombm and other coagulation proteinases [5] proxades an opportunity to develop rigorous assay procedures for m&vidual coagulation protemases and to study the determinants for specificity of these proteinases. Considerable confusion has arisen from development of assay procedures m the absence of appropriate kinetic data and has been discussed previously [6,7]. Specificity of thrombm toward tri- and tetrapepude p-nitroanihde substrates is not expected to be absolute because physiologically important action is known to occur on several protein substrates with considerable variation in the amino acids at the P2 and P3 positions [8,9] (Fig. 1). Several proteolytlcally modified forms of thrombm

540 Substte composmons m proteins cleaved by thrombm Pt Arg(16), Lys(2)

I'2 Pro(10), Ile/Val(4), Gly(3), Ala(2), Met(l), Thr(l)

I'3 Iie/Leu/Val(5), Thr/Ser(5), Gly(2), Arg(2), Gin/Ash(2), Aia(1), Tyr(l), Met(l)

V4 lle/Leu/Vai(7), Gly(5), Gln/Asn(2), Phe(1), Pro(l), Asp(l), Ser(l), Arg(l) Fig 1 Thrombm specificity mferences from protein substrate cleavage by thrombm Data are from Blomback [8]

extubit very different actlvtues toward various protein substrates and mhibitors [10,11] but not toward low molecular weight argmlne esters and andides [12], adding an additional dimension to considerations of thrombm specificity. Although the approach taken here is not an opumal investigation of active-site topography, due to the use of substrates not specifically prepared for tlus purpose, extensive mformatton about thrombm speclfictty ts evident in the data and provades a basts for understanding thrombm specificity. These investigations provide klnettc parameters for bovine thrombm and 24 commercially available peptide p-nitroanthde substrates Dunng these studies several sources of systematic error have been ehnunated which are related to adsorpUon loss as a result of the low concentraUons of protemase required for practical lmttalvelocity determinations. Materials and Methods

p-N l t r o p h e n y l - p - g u a m d i n o b e n z o a t e - H C1 (NPGB) was obtained from ICN Pharmaceuucals, Inc., Cleveland, OH Bz-LArg-pNA was obtained from Bachem, Torrance, CA. Tos-Gly-LPro-LArgpNA and Cbz-Gly-LPro-LArg-pNA (both designated Chromozym TH), Tos-Gly-LPro-LLys-pNA (Chromozym PL), CBz-LVal-GIy-LArg-pNA (Chromozym TRY), and Bz-LPro-LPhe-Arg-pNA (Chromozym PK) were provaded by Pentapharm Ltd., Basel, Switzerland, and Boehringer Mannhelm Blochermcals, Tutzing, F,R.G. CH3SO2DLeu-GIy-LArg-pNA was provaded by Pentapharm Ltd. Bz-LPhe-LVal-LArg-pNA was provided by Peptide Research Institute, Osaka, Japan The remmnder of the substrates used for this investigation (with S-**** notation) were provaded

by AB Kabl, Molndal, Sweden. Poly(ethylene glycol) (PEG) 6000 was obtained from J.T Baker Chermcal Co., Pl'ulhpsburg, N J, PEG 20 000 from Sigma Chemical Co., St. Louis, MO, and bovine trypsin from Wortlungton Blochermcal Corp., Freehold, NJ. The salts and buffers were of analytical reagent grade. Flbnnogen was prepared from bovine plasma by the procedure of Straughn and Wagner [ 13]. Bz-Arg-pNA was dissolved m 1% dimethylformamlde. All of the other peptide p-mtroanlhde substrates were dissolved In deionlzed water that had been adjusted to pH 4 with hydrochloric acid. Substrates stored at - 2 0 ° C showed no hydrolysis after periods longer than 1 year Stereochenucal purity with respect to the PI residue of six of the p-nltroandtde substrates was determined by trypsin hydrolysis. Substrates were dissolved in 0.1 M NaC1/0.01 M Hepes/0.1 M Tns-HC1/0.1% PEG 6000 at approx. 100 /xM. Concentrated bovine trypsin was added to each substrate solution (change in volume was 0 2%) and after incubating at 37°C for at least 2 h absorbance measurements were made at 342.0 nm (the lsosbestlc wavelength) and 405 nm. Substrate and product concentrations were determined by using the appropriate extinction coefficients (see below) The mean extent of hydrolysis was 99.4%, indicating all L-P1 aminoacid residues Bovine thrombm was prepared as described by Owen et al. [14], a modification of the procedure of Lundblad [15] Relative amounts of a- and fl-(degraded) thrombm were determined by mtegrauon of areas from sodmm dodecyl sulfate (SDS) polyacrylarmde electrophoresls gels [16] scanned at 564 nm. Protein was stained with Coomassxe brdhant blue, G-250 [17].

541

concentrations by

Sample preparation for determmatton of peptlde p-nltroamhde hydrolyszs rates

Thrombin stock solutions were centrifuged to remove aggregated protein and were used at a concentration of approx. 1 m g / m l . Active-site titratlons were performed using a Cary 219 double-beam spectrophotometer and a modfflcaUon of the procedure of Chase and Shaw [18] m winch 0.1 M NaC1/0.1 M Hepes, p H 8 3, was used m place of the veronal buffer in the original procedure. The production of p-nltrophenol was measured at 410 nm. The same volume of the thromb m solution used for the actwe-slte titration was added to buffer soluuon in the absence of N P G B to deternune the correction to the absorbance at 410 nm due to the turbi&ty of the thrombin solution Actlve-s~te Utratmns were performed five times for preparation B-a-1 and two Umes for preparaUons B-a-2 and B-a-3 with two to three rephcates each ume.

For the chromogenic substrate hydrolysis rate measurements, thrombm stock solutions were d~luted mto 0.5 M NaC1/0.01 M Hepes/0.01 M Tns-HC1/0.1% P E G 6000, p H 7.8, to give final concentrations between 27 nM and 62 /~M. The dilutions were made into polypropylene titration vessels (Radiometer America, Westlake, OH) winch had been filled with 0.1% P E G 20 000, drained and oven dned to coat the surface with poly(ethylene glycol). Reaction velocmes were determmed by adding the diluted thrombin ahquot to thermostatted polystyrene cuvettes (Walter Sarstedt, Princeton, N J). Compositions of the substrate solutions are noted in the tables. The final thrombin concentraUons vaned from 0.2 to 250 nM. P E G 6000, 0.1% ( w / v ) was included in the assay soluuons to prevent loss of enzyme due to adsorption

Thrombln btoassay

Initial-velocity determmatlons

Clotting actw~ty of the thrombm preparauons was deternuned using the procedure of Fenton and Fasco [19]. A reference curve was prepared using Lot J, US Reference Thrombin and was fit to an arbitrary power curve ( y = ab x) with the parameters determined by linear regression on the logarlthnuc form of the equatton. Correlation coefficients using tins curve to represent the relationship between clotting t~me and t h r o m b m concentration were greater than 0.998 Thrombln samples were assayed m duplicate at four dduuons winch spanned a 100-fold concentrauon range. No systematic variation m the actwlty for the various dilutions was observed, indicating that all thromb m samples behave mdlstmgmshably in tins bioassay and fit the arbitrary power curve. Coeffloents of variation for the bioassay activity deterrmnations were less than 10%. The clotting activlttes are expressed as ' N I H ' u n i t s / m g protein usmg the absorbance at 280 nm and absorption coeffioent of 1 95 m l / m g per cm [14,20]. Corrections of the absorbance measurements for light scattering by the method of Shapiro and Waugh [21] were less than 1%.

Reactions were run at 25°C unless otherwise noted. The rate of peptlde p - m t r o a m h d e hydrolysis was determined from the change in absorbance at 405 nm at a spectral bandwidth of 2.0 nm or less using an absorption coefficient for p-mtroanlline of 9920 M -1 • cm -1 for the 0.1 M NaC1/0.01 M Hepes/0.1 M Tris-HC1 buffer solution, for solutions at other ionic strengths, appropriate extinction coefficients were used [22]. D a t a were transferred d~rectly from the spectrophotometer to a PDP 11/34a digital computer, Digital Eqmpment CorporaUon (Maynard, MA) using a program developed for tins purpose (Scott and Jackson, unpubhshed data). Imtlal velocities were esUmated using a program (Jackson and Carlisle, unpublished data) based on the direct linear plot procedure of Comish-Bowden [23] * For some of the earliest data, initial veloclaes were determined from strip chart recordings using a ruler and pencil. For both methods, at all but the very lowest substrate concentrations, less than 10% of the substrate was hydrolyzed. Substrate concentratlons ranged from approx. 0.5-times K m to

Determlnatwn of thrombm actlve-stte tttratton

* A nuspnnt exasts in Eqn 2 of tbas reference, vlz P~o Is m~ssmg from the numerator of the last term m the equation

542 15-times Km, or the baghest practical concentration (usually limited by solubihty of the substrate) Kinetic parameters were calculated from replicate determinations of the initial velocity Substrate concentration ranges and number of lmtial-veloclty determinations are presented m the tables Substrate concentrations were determined from the dilution of stock solutions and, for solutions greater than 10 ~M, confirmed by measurmg the absorbance of the reaction rmxtures at the lsosbestic wavelength (342.0 nm for 0 1 M NaC1/0.01 M Hepes/0.01 M Tns-HC1) after the reaction was completed. An absorption coefficient at 342 0 nm of 8270 M - 1 . cm-1 was used for the calculations with thas buffer solution. For reaction solutions with ionic strengths other than 0 107 the appropnate lsosbest~c wavelengths and the corresponding extinction coefficients were as deterrmned by Lottenberg and Jackson [22].

Ktnetw parameter determmatwns Kinetic parameters were deterrmned by non-hnear least-squares fitting of the simple MlchaehsMenten equation to the data assuming constant variance at all velocities [24] and the computerbased version of the direct linear plot method [25,26]. Published Fortran programs [27] for these procedures were modified for use with the output files of the data acquisition and reduction programs noted above For the substrates listed in the tables without independent values for the Mlchaehs constant (Krn) and the catalytic constant (kcat), i.e., those for wluch the lughest attainable substrate concentration was significantly below the K,,, the kcat//gm was esUmated from an unwelghted hnear regression fit to the substrate concentration range whach gave a hnear dependence of the velocity. Results

A. Techmcal :ssues Est:matton of thrombm molar concentrattons, btoassay actwtty and distribution of thrombm samples among the various proteolytwally modtfwd forms Active thrombin concentrations are required to calculate the catalytic constant (kcat) from the maximum velocity The three bovine a-thrombm preparations employed m these investigations were

greater than 94% active by active-site titration. The coefficients of variation for the thrombln concentraUons ranged from 0 to 3.8% (mean of 1 8%) for 21 active-site Utrations of thrombln preparations at concentrations from 25 2 to 35.5 #M Overall relative standard deviations for the estimates of kcat are less than 8%. Specific actwlty and distribution of the thrombin between the two forms, a and /3, are as follows. thrombin preparation B-a-2, 4000 N I H u / m g and less than 7% fl-thrombin; B-a-3, 3450 N I H u / m g and less than 14% fl-thrombin; and B-a-l, 3400 N I H u / m g and less than 1.5% flthrombin. Loss of thrombm due to adsorpt:on from stock soluttons and during hydrolys:s rate determmatwns. Loss of thrombm as a result of adsorption to the wails of the vessels into wluch the thrombm was being diluted was the greatest source of systematic error that had to be ehmlnated dunng these investigations. Dilution into poly(ethylene glycol)-coated polypropylene vessels and buffers containing PEG 6000 as described previously [28] ehmlnated the adsorpuon losses entirely. When no competitive adsorbate was included m the buffer solutions as much as 50% loss of enzyme activity was observed When diluted to low concentrations for actual assay enzyme activity may be lost due to adsorption to the cuvette walls and inactivation of the adsorbed enzyme The procedure descnbed by Selwyn [29] was used to investigate possible loss of enzyme activity under the assay conditions employed in this study. The progress curves were supenmposable when plotted as product concentratlon vs. enzyme concentration muluplled by time for thrombm at concentrations between 0 236 and 10.1 nM, indicating stable enzyme activity for more than 10-times the period employed for the imtml-veloclty determinations. All replicate assays also were performed In a defined order such that any time-dependent loss of enzyme activity in the stock enzyme solution would be evident as a trend toward decreasing velocities with time. The absence of systematic differences between replicate velocities for any of the preparations confirmed the stability of the enzyme in the dilution buffer in all experiments Effects of poly(ethylene glycol) on the kmetw

543 TABLE la BOVINE a-THROMBIN KINETIC PARAMETER DEPENDENCE ON POLY(ETHYLENE GLYCOL) (PEG) CONCENTRATION Substrate Tos-Gly-LPro-LLys-pNA (Chromozym PL) Data are for 25 0°C, pH 8 0, 0 10 M NaCI, n = the number of datum points, the range is the substrate concentratton range K m and kca t values below the mean gtve the range for one standard deviation for nonhnear regression deterrmned values

m(M - 1 s- 1)

Reaction conditions

kcat / K

PEG 6000 0 1% PEG (n = 16, 4 88-255 #M) a

1 11 106

0 5% PEG (n = 12, 4 58-186/zM)

1 01 10 6

1 0% PEG (n = 12, 4 97-148/~M)

8 90 105

2 0% PEG (n = 12, 4 62-182/zM)

9 16 105

5 0% PEG (n = 12, 5 15-188 #M)

648 105

100% PEG (n = 12, 5 51-162/~M)

3 74 105

15 0% PEG (n = 14, 5 27-286 ~M)

2 62 105

PEG 20000 l 0% PEG (n = 12, 4 53-190/~M)

8 60 105

5 0 PEG (n = 14, 4 56-334 jaM)

2 90 105

l0 0% PEG (n = 14, 4 39-333 #M)

l 40 105

kcat (s- 1)

23 0 (22 2-23 23 9 (23 3-24 23 4 (22 6-24 24 0 (23 3-24 25 9 (25 4-26 23 5 (21 7-27 24 9 (24 i-25

K m (~ M)

7)

20 6 (17 6-23 6) 23 7 (21 9-25 5) 26 3 (23 5-29 1) 26 2 (23 8-28 6) 40 0 (37 9-42 l) 62 9 (51 7-74 l) 95 2 (87 3-103 0)

24 5 (23 8-25 2) 27 2 (26 6-27 8) 21 5 (19 9-23 l)

28 5 (26 0-31 0) 93 6 (88 0-99 2) 1540 (129 0-179 0)

8) 5) 2) 7) 4) l)

a Data are from Table II, pH 7 8

parameters for thrombm hydrolysts of Tos-Gly-Pro-Lys-pNA and Bz-Arg-pNA. T h e use of P E G 6000 m a s s a y b u f f e r s a n d P E G 20 000 to c o a t t h e s u r f a c e of d i l u t i o n vessels n e c e s s i t a t e d exa r m n a t l o n o f effects o f tins a g e n t o n the l o n e t l c parameters. Hydrolysis of Tos-Gly-Pro-Lys-pNA a n d B z - A r g - p N A w e r e e x a m i n e d as a f u n c t i o n of p o l y ( e t h y l e n e glycol) c o n c e n t r a t i o n f o r P E G 6000 a n d P E G 2 0 0 0 0 , T a b l e s Ia a n d Ib, r e s p e c t i v e l y . W i t h T o s - G l y - P r o - L y s - p N A , kcat//Km d e c r e a s e d 4 - f o l d b e t w e e n 0.1 a n d 15% P E G 6000 a n d 8-fold b e t w e e n 1 a n d 10% P E G 2 0 0 0 0 , d u e e n t i r e l y to a n i n c r e a s e in K m in b o t h cases. I n c o n t r a s t to tins s i t u a t i o n , w i t h Bz-Arg-pNA a n 8-fold i n c r e a s e in K m a n d a 2 - f o l d d e c r e a s e in kca t w e r e n o t e d w i t h P E G 6000, a n d a 14-fold i n c r e a s e m K m a n d a 2 - f o l d d e c r e a s e in kca t w i t h P E G 2 0 0 0 0 . T h e r e are n o s i g n i f i c a n t d i f f e r e n c e s b e t w e e n the k i n e t i c

p a r a m e t e r s for 0.1 a n d 1% p o l y ( e t h y l e n e glycol) a n d t h u s the p o l y ( e t h y l e n e glycol) a p p a r e n t l y is a c t i n g o n l y as a c o m p e t i t i v e a d s o r b a t e at t h e s e c o n c e n t r a t i o n s . T h e c o n c e n t r a t i o n o f P E G 6000 u s e d r o u t i n e l y m a s s a y b u f f e r s for p e p t i d e p m t r o a m h d e s u b s t r a t e s (0.1%) has t h u s n o effect o n the reactions being investigated.

B Substrate selecttvtty of thrombm Kmenc parameters. I O n e t i c p a r a m e t e r s for the 24 s u b s t r a t e s are p r e s e n t e d in T a b l e II for s e v e n arglmne and one lysme p-mtroanihde with proline o r p r o h n e h o m o l o g s in the Pz p o s m o n ; in T a b l e I I I for six a r g m m e p - n i t r o a m h d e s w i t h g l y c m e at P/; in T a b l e I V for five s u b s t r a t e s w i t h e i t h e r a r g i n i n e o r l y s m e at the PI p o s i t i o n a n d p h e n y l a l a n m e at P2, a n d m T a b l e V for s u b s t r a t e s w i t h a r g m l n e o r lyslne at PI a n d e i t h e r v a h n e o r l e u c m e

544 TABLE Ib BOVINE a-THROMBIN KINETIC PARAMETER D E P E N D E N C E ON POLY9ETHYLENE GLYCOL) (PEG) CONCENTRATION Substrate Bz-LArg-pNA Data are for 25 0°C, pH 8 0, 0 10 M NaC1, n = the number of data points, the range is the substrate concentratton range g m and kca t values below the mean gwe the range for one standard devmuon for nonlinear regression determined values Reactaon conchtaons

kca t//K m (M - 1 s - I )

kca t (s 1)

PEG 6000 0 1% PEG (n = 13, 28 7-2870 ~tM)

6 27 102

0 0714 (0 0699-0 0 0721 (0 0707-0 0 0546 (0 0533-0 00409 (0 0384-0 0 0327 (0 0284-0

1 0% PEG (n = 14, 29 2-2920/~M)

4 20 102

5 0% PEG (n = 12, 60 8-3040 pM)

1 86 l02

100% PEG (n = 12, 63 6-3180 pM)

7 29 101

15 0% PEG (n ffi 9, 131-3280/tM)

3 40 l01

PEG 20000, 0 I M NaC1, pH 8 0 1 0% PEG (n = 14, 28 7-2870 #M)

3 11 l02

50% PEG (n •10, i15-2870 pM)

5 35 101

l0 0% PEG (n = 9, 115-2870/~M)

2 27 101

at P2. The k c a t / / K m values ranged from 33 M - 1 . s-1 for PyrGlu-Phe-Lys-pNA to 1 • 108 M - l . s-1 for oPhe-azetidinecarboxyhc acld-Arg-pNA. For those substrates for wluch both kinetic parameters were deterrmned, the K m values ranged from 0.448 to 350 p M and the kca t values ranged from 0.0526 to 154 s -1. The relatlonstups between k c a t / / K m , kca t and K m and substrate structure m order of decreasing kcat/K m are dlustrated m the bar graphs of Fig. 2. The K m for oPhe-Plp-Arg-pNA m Table II ts the weighted mean from three sets of velocity vs substrate concentration data, one wxth each of the thrombin preparations. The kca t values are mean values from preparations B-a-2 and B-a-3; the kca t for B-a-1 was 10% lower than this mean. The K m value in Table III for Tos-Gly-Pro-Arg-pNA ~s the weighted mean from eleven separate sets of velocity vs. substrate concentration deternunat~ons. Nine of these sets were w~th the B-a-1 preparation, one set with B-a-2 and one set with B-a-3. The kca t values are means of three values. Kinetic parame-

K m (p M)

- -

0370)

114 (103-125) 172 (160-184) 294 (271-317) 561 (464-658) 962 (683-1241)

0 0649 (0 0624-0 0674) 0 0674 (040558-0 0790) 0 0356 (0 0332-0 0380)

209 (180-238) 1 260 (790-1 730) 1 570 (1 360-1 780)

0729) 0735) 0559) 0434)

ters for all other substrates were deterrmned with preparation B-a-1.

The effects of NaCI and CaCI e on the hydrolysis of Tos-Gly-Pro-Arg-pNA Kinetic parameters for t h r o m b m action on Tos-Gly-Pro-Arg-pNA as a function of NaCI concentration are presented m Table VI; the values for 0.1 M NaC1 are taken from Table II. The constant, keat/K m, decreased 4-fold, due to a larger increase m K m than m kca tCaC12 concentrations were vaned between 0 and 33 mM, with the Ionic strength being kept constant at 0.107 by adding NaCI to the reacuon solutions (Table VII). CaC12 had no significant effect o n g m when NaCI was present to maintain constant ~onic strength; kca t decreased w~th increasing CaC12 concentrations, to 70% of its value m the absence of CaC12. At 167 m M CaCI 2, eqmvalent to 0.5 M NaCI, a s~gmflcant effect was observed on K m that was 3-fold greater than the effect of NaCI.

The effects of p H on the hydrolysts of pepttde p-nttroamhde substrates. The p H dependence of

545 TABLE II SUBSTRATE SELECTIVITY BOVINE a-THROMBIN Vamable P3-P4 X-X.'Pro'-(Arg/Lys)-pNA Data are for 2500C, pH 7 8, 001 M Trls-HCl/001 M Hepes/0 10M NaCI/0 1% PEG 6000 n = the number of datum points, the range is the substrate concentrauon range kca t and K m values below the mean gtve the range for one standard deviation for nonhnear regression deternuned values Substrate

kcat/Km(M-l

H-x)Phe-Aze-LArg-pNA (n = 14, 0 421-107 pM) S-2388 a

1 07 108

47 7 (46 6-48 8)

0 448 (0 388-0.508)

H-I)Phe-LPlp-LArg-pNAb (n = 40, 0 538-65 3/tM) S-2238 a

6 56

98 4 (96 4-100)

i 50 (I 40-1 60)

H-I)Ile-LPro-LArg-pNA (n = 14, 0405-97.0 pM) S-2288 a

6 18 107

74 0 (72 1-75 9)

1 20 (1 06-1 34)

H-DVal-LPro-LArg-pNA (n = 14, 0422-104 pM) S-2234 a

4 45

88 8 (86 3-91 3)

1 99 (1 75-2 23)

Tos-GIy-LPro-LArg-pNAc (n = 130, 0 96-187 pM) Chromozym TH a

2 79 107

102 (98 0-106)

3 61 (3 46-3 76)

PyrGlu-LPro-LArg-pNA (n = 14, 13 6-492 pM) S-2366 a

4 06 106

Cbz-Gly-LPro-LArg-pNA (n ffi 18, 0.562-95 2 pM) Chromozym TH a

3 31 106

83 0 (80 8-85 2)

25 0 (23 4-26 6)

Tos-Gly-LPro-LLys-pNA (n = 16, 4 88-255 #M) Chromozym PL a

1 11-106

23 0 (22 2-23 8)

20 6 (17 6-23 6)

a b c d

s-1)

10 7

10 7

kcat (s-I)

154 (151-157)

g m (pM)

38 0 (34 9-41 1)

Kabl Peptlde Research, Molndal, Sweden Value given is from three sets of mdependently deternuned velocity vs substrate concentration curves Value gwen ~s from 11 sets of independently determined velocity vs substrate concentration curves Pentapharm Ltd, Basel, Switzerland, and Boehnnger Marmhelm GmbH, Tutzmg, F R.G

t h r o m b m action on T o s - G l y - P r o - A r g - p N A a n d T o s - G l y - P r o - L y s - p N A is s u m m a r i z e d m Tables V I I I a and V I I I b , respectively. F o r b o t h substrates, K m values decrease and kca t values increase with increasing p H. T h e increase in rate b e t w e e n p H 5 a n d 8 was f o u n d to reflect b e h a w o r suggesting an ' a p p a r e n t p K ' of 7.5 f r o m plots of log kcat/Km vs. p H [30] an d f r o m direct e x a m i n a t i o n of the p H d e p e n d e n c e for the d a t a f r o m b o t h substrates norm a h z e d to the kcat//Km values at p H 7.8 (Fig. 3).

Effect of temperature on the hydrolys,s of pepttde p-mtroamhde substrates The t e m p e r a t u r e depend e n c e of the hydrolysis of o P h e - P i p - A r g - p N A a n d

T o s - G l y - P r o - A r g - p N A , two substrates with high kca t values, and C b z - V a l - G l y - A r g - p N A , a substrate with a low kcat, was e x a m i n e d at 15 an d 3 7 ° C as well as at 25°C. T h e kinetic p a r a m e t e r s are p r esen t ed in T a b l e I X (the d a t a for 2 5 ° C are taken f r o m Tables II an d III). T h e a c t i v a t i o n energtes for each of these substrates were calculated f r o m the kca t values an d the A r r h e n m s eq u at i o n . Plots of log kca t vs. 1 / T ( K ) were h n e a r and A r r h e n i u s activation energaes of 11.2, 12.4 an d 14.3 k c a l / m o l for T o s - G l y - P r o - A r g pNA, DPhe-Pip-Arg-pNA an d C b z - V a l - O l y - A r g pNA, respectively, were o b t m n e d .

546 TABLE III SUBSTRATE SELECTIVITY BOVINE a - T H R O M B I N Variable P3-P4 X-X-GIy-Arg-pNA Data are for 25 0°C, pH 7 8, 0 01 M T n s - H C I / 0 01 M H e p e s / 0 10 M N a C 1 / 0 1% PEG 6000 n = the number of datum points, the range ~s the substrate concentration range K,~ and kca t values below the mean g#ve the range for one standard devtatmn for nonhnear regression deterrmned values Substrate

kca ' / K m ( M - ] s - ])

kca t ( s - 1)

K m (/aM)

Bz-PyrGlu-Gly-LArg-pNA (n = 10, 11 3 - 3 1 8 / a M )

441 105

284 (27 6 - 2 9 2)

CH3SO2-DLeu-GIy-LArg-pNA (n = 14, 23 1-462/aM) (Pentapharrn) b

2 49 l0 s

42 0 (41 1-42 9)

Bz-Llle-LGlu(lhp)-Gly-LArg-pNA c (n = 10, 22 5-305 jaM) S-2337 a

6 57 104

2 63 (2 5 0 - 2 76)

40 0 (34 0 - 4 6 0)

Cbz-LVaI-Gly-LArg-pNA (n = 13, 11 2 - 2 2 9 / a M ) Chromozym T R Y b

4 48 104

3 24 (3 13-3 35)

72 3 (66 7 - 7 7 9)

Bz-Llle-LGlu-GIy-LArg-pNA c (n = 18, 9 15-524/aM) S-2222 a

1 69 104

0 941 (0 9 2 1 - 0 961)

55 6 (51 9 - 5 9 3)

PyrGlu-Gly-LArg-pNA (n = 4, 53 4--210 /aM) S-2444 a

4 44 10 3

645 (59 9 - 6 9 1)

S-2405 a

168 (160-176)

--

__ d

a Kabl Pepude Research, Molndal, Sweden b Pentapharm Ltd, Basel, Swatzerland c An lmtml 'lag' prior reachmg steady-state was not apparent vath S-2337, whereas a 'lag' was observed wRh S-2222 d Km was greater than 210/aM, and the velocRy vs substrate concentrataon dependence hnear.

i m CH3S~-O Leu GL~ L ~ ~ m m m J m m ~ l ~ a m ~ ~z L ILE L ~U(PIP) ~ y - L ~ G ~ m ~ l m J l H cot/~ ~

I ]/MIS~CI

X

CBI-L V~-GLy-L ARe~ANEMilUl I

eZ-L-ICe L eLU ~ , L A ~ F O B

~-!L~-,_-~-L-,,, ~-_~-~..~,,~L

h r o L I K m 41 IMxSEC]

BZ-L let-: ~.u(Pl~)-e.v-L-~ C~ C W. ~ eb oo

t~o oo I~col~ I[/SEC~

t ~ oo

t~,o oo

i ~ co

=1~ oo

L ~G

BZ-t.-ILe-L i~OLr-L A~¢

o oo

s' oo

ib oo

~ oo

a~ o~ fs oo k~Q~ I I / S E C I

io ~o

~ oo

uID oo

~ o~

190 00

two 00

I'~0 ~

I ~ 00

W-O.~e-L.-h e - L - ~ i M-~-JLt'L"~O'L-X" •

8z L-ILI-L-GLu(P~P)-6LY C I ~

PveO.u ~L,-L AA6

K ~ {M] CROMOLRR)

0 O0

2b 0 0 ~ o

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0'0 00 lb0 00 IM]CROMOLARI

547

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r

1

H-O-V~-L-LE~ L

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~b oo

~

eb o~

~b oo

tbo

oo

Ao oo

tto oe

i~o o~

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~ °0 vv~ LLLLE£Z=LL L~C$I

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t~o oo

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Fig 2 Substrate selecUxaty and pept]de structure A Variable P3-P4 X-X-'Pro'-(Arg/Lys)-pNA B Variable P3-P4 X-X-GIy-Arg-pNA C Variable P~-P4 X-X-Phe-(Arg/Lys)-pNA D Variable P3-P4 X-X-(Val-Arg/Leu-Lys)-pNA

TABLE IV S U B S T R A T E SELECTIVITY BOVINE a - T H R O M B I N Variable P3-P4 X-X-Phe-(Arg/Lys)-pNA Data are for 25 0°C, pH 7 8, 0 01 M T n s - H C I / 0 01 M H e p e s / 0 10 M NaC1/0 1% PEG 6000 n = the number of datum points, the range ~s the substrate concentratmn range K m and kca t values below the mean gwe the range for one standard dewatmn for nonlinear regression deternuned values Substrate

k c a t / K m ( M - 1 s - 1)

kca t ( s - I)

g m (#M)

H-DVal-LPhe-L~g-pNA (n = 10, 13 7-311 pM) S-2325 a

1 74 105

22 4 (21 4 - 2 3 4)

129 (117-141)

H-DPro-LPhe-LArg-pNA (n = 12, 7 09-468 # M ) S-2302 a

9 93 104

12 5 (11 7-13 3)

126 (108-144)

H-DVaI-LPhe-LLys-pNA (n = 4, 110-289 # M ) S-2390 a

1 84 103

Bz-LPro-LPhe-LArg-pNA c (n = 8, 46 1-439 # M ) Chromozym PK o

4 83 102

PyrGlu-LPhe-LLys-pNA (n = 4, 324-599 # M ) S = 2403 a

3 31 101

a b c d •

--

0 0526 (0 0 4 8 5 - 0 0567) --

Kabt PepUde Research, Molndal, Sweden Km was greater than 290 pM, and the velocity vs substrate concentraUon dependence linear An ~mtml 'lag' prior to reacbang the steady-state was apparent Pentapharm Ltd, Basel Switzerland and Boehnnger Mannhelm GmbH, Tutmng, F R G K m was greater than 600 #M, and the velocity vs substrate concentration dependence hnear

__ b

109 (88-130) -- ¢

548 TABLE V SUBSTRATE SELECTIVITY BOVINE a - T H R O M B I N Variable P3-P4 X-X-(Val-Arg/Leu-Lys)-pNA Data are for 25 0°C, p H 7 8, 0 10 M NaCI n = number of d a t u m points, the range is the substrate concentration range K m and kca t values below the mean gjve the range for one standard deviation for nonhnear regression deterrmned values Substrate

k c a t / / K m ( M - 1 S-- 1)

k c a t ( S - 1)

Bz-L(mtro)Phe-LVal-LArg-pNA (n = 20, 11 3 - 6 7 0 / ~ M ) S-2160 a

2 13 106

37 5 (35 7 - 3 9 3)

17 6 (15 0 - 2 0 2 )

Bz-LPhe-LVal-LArg-pNA (n = 29, 14 8-131 # M ) Protein Research Foundatton b

5 34 105

37 8 (35 6 - 4 0 0)

70 8 (62 3-79 3)

H-DVal-LLeu-LArg-pNA (n = 8, 78 3 - 4 6 6 / z M ) S-2266 a

5 35 I 04

18 7 (16 5 - 2 0 9)

H-DVaI-LLeu-LLys-pNA (n = 4, 95 2-436/*M) S-2251 a

7 99 102

Bz-LArg-pNA d (n = 13, 28 7-2870/~M)

6 27 102

a b c d

K m (# M)

350 (273-427)

--

0 0714 (0 0699-0 0729)

__ c

114 (103-125)

Kabl PepUde Research, Molndal, Sweden Peptlde Institute, Protein Research Foundation, Osaka, Japan Km was greater than 436 ~aM, and the velocity vs substrate concentration dependence hnear p H 80, 0 1 M NaC1/0 01 M H e p e s / 0 1% PEG 6000

T A B L E VI BOVINE a - T H R O M B I N K I N E T I C P A R A M E T E R D E P E N D E N C E O N IONIC S T R E N G T H Substrate Tos-GIy-LPro-LArg-pNA (Chromozym TH) Data are for 25 0°C, p H 7 8, 001 M T n s - H C I / 0 0 1 M H e p e s / 0 1% PEG 6000, NaCI as speofled n = the number of d a t u m points, the range is the substrate concentrauon range K m and kea t values below the mean gtve the range for one standard devaatton for nonhnear regression deterrmned value Reaction c o n d m o n s

kcat/Km ( M - ]

0 02 M NaCI (n = 9, 5 19-78 9 ~M)

5 76 107

91 8 (89 8-93 8)

1 59 (1 29-1 89)

0 05 M NaCI (n = 10, 2 53-87 2/~M)

2 91 107

96 7 (93 9 - 9 9 5) 102

3 33 (2 94-3 72) 361

lO1 (99 0-103)

5 39 (4 74-6 04)

0 1 M NaC1 a 0 2 M NaC1 (n =10, 3 3 3 - 6 6 6 / ~ M )

2 7 9 107 1 87 107

s-I)

kca t ( s - 1 )

K m (/~M)

0 5 M NaCI (n = 23, 1 98-99 2/LM)

1 79 107

108 (106-110)

6 02 (5 6 1 - 6 43)

1 0 M NaC1 (n = 9, 4 9 8 - 8 1 2/~M)

1.48-107

124 (119-129)

8 40 (7 10-9 70)

a Data from Table II

549 T A B L E VII BOVINE a - T H R O M B I N K I N E T I C P A R A M E T E R D E P E N D E N C E O N CaCi 2 C O N C E N T R A T I O N Substrate Tos-GIy-LPro-LArg-pNA (Chromozym TH). Data are for 25 0°C, p H 7 8, 0 01 M T n s - H C 1 / 0 01 M Hepes, NaCi vanable, 0 1% PEG 6000 n = the number of d a t u m points, the range is the substrate concentration range K m and kca t values below the mean give the range for one standard devmtlon for nonhnear regression deterrmned values Reaction c o n d m o n s

kca t / K i n ( M - l s - I )

I = 0 107 no Ca a l mMCa (n = 9, 2 4 1 - 4 4 8 p M )

kca t ( s - 1)

Km (btM)

2 79 107

102

2 30 107

99 4 (94 2-105)

4 33 (3 57-5 09)

10 m M Ca (n = 10, 2 41-44 8 pM)

2 58 107

97 4 (95 5 - 9 9 3)

3 77 (3 5 2 - 4 02)

33 m M Ca (n = 10, 2 41-44 8 pM)

I 83 107

73 1 (71 0 - 7 5 2)

400 (3 6 2 - 4 38)

•=05 no CaC12, 0 5 M NaCI b (n = 23, 1 9 8 - 9 9 2 p M )

1 79 107

167 m M Ca (n = 9, 2 41-44 8 p M )

421 106

108 (106-110) 726 (70 6 - 7 4 6)

3 61

6 02 (5 6 1 - 6 43) 17 2 (16 1-18 3)

a Data are from Table II b Data are from Table VI 2G

Discussion 18

Issues affecting the accuracy of the kmetw parameter estimates Large systematic errors m enzyme concentration were observed as a result of adsorption of thrombm to vessels into which high ddutlons were being made for assay. Concentrated solutions used for actwe-slte tttratlon were stable for more than 1 week at 0 - 4 ° C , indicating that the thrombin itself was not becoming inactive. Adsorption loss was ehrmnated by the inclusion of P E G 6000 m the dilution buffers and by precoatmg the surfaces of the polypropylene vessels with P E G 20 000. Under these conditions thrombln at 10 nM and lower concentrations has been found to be stable for 18 h at 25°C. In view of the observation that

16

14 E "--12 -at

N

08

06 0d

/ o

02

6

7

1'o

8

pH

Fig 3 pH dependence of kcat/Km for Tos-Gly-Pro-Arg-pNA and Tos-Gly-Pro-Lys-pNA Data are normahzed to the value for kcat/g m at pH 7 8 All data are from Tables V i l l a and VIllb

550 TABLE VIIIa BOVINE a-THROMBIN KINETIC PARAMETER DEPENDENCE ON pH Substrate Tos-Gly-LPro-LArg-pNA(Chromozym TH) Data are for 25 0°C, pH as specified, 0 1 M NaCI n = the number of datum points, the range is the substrate concentration range K m and keat values below the mean give the range for one standard devaation for nonhnear regression deterrmned value Reaction condmons

kcat//K m (M- 1 s- l)

pH 500 a ( n = l i , 188-409pM)

181 105

29 2 (26 9-31 5)

pH 5 50 b (n = 10, 34 1-409/~M)

9 53 105

79 3 (760-826)

83 2 (724-940)

pH600 b (n ffi 12, 5 50-297 pM)

1 13 106

68 8 (65 9-71 7)

61 2 (54 3-68 1)

pH 6 50 (n = 10, 5 36-129 pM)

3 38 106

88 0 (83 5-92 5)

26 0 (22 2-29 8)

pH 6 75 (n = 12, 4 99-92.4 pM)

5 39 106

91 6 (88 5-947)

17 0 (15 2-18 8)

pH 7.00 (n = 10, 0 924-16 3 pM)

8 91 106

96 4 (84 5-108)

10 8 (8 30-13 3)

pH 7 25 (n = 12, 200-55 3 #M)

1 25 107

101 (97 0-105)

8 08 (7 14-9 02)

pH 7 50 (n = 12, 1 50-56 5 pM)

1 83 107

101 (88 0-104) 102

5 52 (5 03-6 01) 3 61

pH 7 80 c pH 8 50 (n ffi 10, 0 961-16 0 pM)

2 79 107

kca t (S- 1)

K m (#M)

161 (130-192)

3 25 107

108 (103-113)

3 34 (2 87-3 81)

pH 900 (n ffi 12, 0 698-29 2/~M)

4,44 l0 T

127 (123-131)

2 86 (244-3 28)

pH 9 50 (n = 11, 0 781-28 7/.tM)

2 65 107

128 (121-135)

4 82 (3 87-5 77)

a Buffered with 0 01 M sodmm citrate b Buffered wath 0 02 Pipes c Data are from Table II

p o l y ( e t h y l e n e glycol) at h i g h c o n c e n t r a t i o n s pert u r b s the r e a c t i o n s o f s o m e e n z y m e s , p a r t i c u l a r l y t h o s e t h a t self a s s o c i a t e [31] a n d m a y alter b o t h t h e M i c h a e h s c o n s t a n t a n d kea t ( T a b l e s I a n d II), t h e i n f l u e n c e o f p o l y ( e t h y l e n e glycol) s h o u l d b e m v e s u g a t e d m s y s t e m s m w h i c h ~t is e m p l o y e d . N o i n c o n s t a n t effects o f p o l y ( e t h y l e n e glycol) h a v e

b e e n o b s e r v e d In these i n v e s t i g a t i o n s , m c o n t r a s t to o n e o b s e r v a t i o n m a d e w i t h h u m a n t h r o m b m [32]. Our practice of confirrmng concentrations of s u b s t r a t e s o l u t i o n s b y m e a s u n n g the s o l u u o n abs o r b a n c e at the l s o s b e s t i c w a v e l e n g t h led to the identification of soluuon compositmn dependent

551 TABLE VIIIb BOVINE a-THROMBIN KINETIC PARAMETER DEPENDENCE ON pH Substrate Tos-Gly-LPro-LLys-pNA (Chromozym PL) Data are for 25 0°C, pH as specified, 0 1 M NaCI n = the number of datum points, the range is the substrate concentraaon range K m and keat values below the mean ~ve the range for one standard devaaaon for nonhnear regressmn deterrmned value Reactton condmons

k c a t / g m (M-1 s-1)

pH600 (n = 12, 16 2-686 #M)

2 59 104

kca t (s-1)

8 22 (8 15-8 29)

K m (#M)

317 (263-371)

pH650 ( n = 12, 35 4-686 #M)

7 76 104

12 4 (12 0-12 8)

pH 700 (n =14, 924-265 #M)

270 105

19 4 (18 9-19 9)

71 7 (66 7-76 7)

pH 7 25 (n = 12, 35 4-686 #M)

3 58 105

194 (18 9-19 9)

54 3 (50 0-58 6)

pH 740 (n = 12, 2 96-190 #M)

5 62 105

260 (24 9-27 1)

462 (40 9-51 5)

pH 7 50 (n = 16, 9 70-196 #M)

5 33 105

25 1 (24 6-25 6)

47 2 (44 5-49 9)

pH 7 65 (n = 12, 2 92-191 #M)

7 76 105

27 4 (26 5-28 3)

35 3 (32 0-38 6)

pH 7 80 (n = 16, 4 88-255 #M)

11 106

23 0 (22 2-23 8)

20 6 (17 6-23 6)

pH 800 (n = 14, 9 70-144 #M)

15 106

22 6 (22 0-23 2)

19 7 (17 9-21 5)

pH 8 25 (n =11, 3 31-108 #M)

24 106

25 3 (24 6-26 0)

20 4 (18 8-22 0)

( n = 25, 3 50-146/~M)

26 106

264 (25 7-27 1)

209 (19 1-22 7)

pH 8 75 (n = 12, 3 51-107 #M)

51" 106

25 3 (24 8-25 8)

16 8 (15 8-17 8)

pH 900 (n = 14, 8 78-143 #M)

51 106

16 7 (15 1-18 5)

25 1 (24 4-25 8)

pH 9 50 (n = 11, 8 78-180 #M)

29 106

22 5 (22 3-22 7)

17 4 (16 9-17 9)

pH 10 00 (n = 12, 7 32-166 #M)

03 106

22 5 (22 !-22 9)

21 9 (20 7-23 l)

160 (146-174)

pH 8 50

552 TABLE IX BOVINE a-THROMBIN KINETIC PARAMETER D E P E N D E N C E ON TEMPERATURE Data are for 25 0*C, pH 7 8, 0 01 M Tns-HCI/0 01 M Hepes/0 10 M NaC1/0 1% PEG 6000 n = the number of datum points, the range ~s the substrate concentraUon range Kra and kca t values below the mean gtve the range for one standard dev~auon for nonhnear regression deterrmned values Reaction condmons

kcat//Km ( M - I

s-1)

Substrate Tos-Gly-LPro-LArg-pNA (Chromozym TH) 15°C (n =16, 0633-107 ~tM) 206 107 25oc a 37°C (n ~ 16, 0 956-90 8 ~tM)

2 79 107 3 68 107

Substrate H-I)Phe-Ptp-LArg-pNA (S-2238) 15°C (n = 16, 0 635-117 ~tM) 5 29 107 25oc a 37°C (n = 15, 1 06-103 ~M)

6 56 107 7 08 107

Substrate Cbz-LVaI-Gly-LArg-pNA (Chromozym TRY) 15°C (n = 14, 274-280 ~aM) 3 58 104 25oc b (n = 13, 11 2-229 pM)

4 48 104

37°C (n = 14, 7 02-279/LM)

4 90 104

kea t (S-1)

K m (/~M)

53 3 (52 3-54 3) 102 204 (198-210)

2 59 (2 36-2 82) 3 61 5 55 (4 86-6 24)

440 (43 1-44 9) 98 4 196 (193-199)

0831 (0 729-0 933) 1 50 2 77 (2 60-2 94)

1 61 (1 48-1 74) 3 24 (3 13-3 35) 9 03 (8 44-9 62)

450 (32 2-57 8) 72 3 (66 7-77 9) 184 (161-207)

" Data from Table II b Data from Table III

shifts m the spectra of the pepude p-nltroamhdes and m p-nitroanlhne. The results of deterrmnation of the extraction coefficients for the peptlde pnitroandides and p-nitroandme are reported elsewhere [22], but the results of that study were incorporated into the determination of the reaction velocities from which the kinetic parameters reported here were calculated. The observation that Michaelis constants as low as 0.5 /~M exast for the best thrombm substrates made determination of accurate estimates for the imtml velocities for these substrates problematmal as total hydrolysis of 1 /~M peptlde p-mtroanlhde gtves rise to an absorbance change at 405 nm of only 0.0099 absorbance umts in 1 cm cells. Bias m mmal-velocity estimation under such condiuons was amehorated by deterrnming the imtial velooues by fitting the integrated M~chaehs-Menten equaUon to the progress curves The nonparametn c stat~sUcal method proposed by Comish-Bowden

[23] and independently evaluated by Nlmmo and Atkins [33] was employed. Preliminary mvesUgations of the shapes of the progress curves using the program ' P R O C U R A ' of Duggleby and M o m s o n [34] and lmual-velocity studies using isolated peptlde products (Lottenberg and Jackson, unpublished observations) indicated that product mhlbiuon was competitive, and thus this algonthm ~s appropriate for determination of the lmual velooues. The two procedures used for fitting velocity versus concentration data to the Michaehs-Menten equation have been shown [24,35-37] to provide more accurate estimates for the lonettc parameters than the routinely employed unwelghted fits to hnear transformations of the Mlchaehs-Menten equaUon [37]. The nonlinear least-squares method [35,37] assumes neghglble error m the substrate concentration and a Gaussmn dxstnbuUon of the errors in the velocity The non-parametric method

553 of Eisenthal and Cormsh-Bowden avoids these assumptmns and is less sensitive to outhers [25]. For those substrates for which values for kca t and K m as well as kcat/K m are reported m the tables, the klneuc parameters determined by either method were never sigmficantly different. Tins is conslstent w~th a Gaussmn distribution of the errors of the velocity deternunaUons for these substrates and thrombm, and imphes that the nonlinear least-squares method provades appropriate est~mates for the lanet~c constants. Plots of the differences between the observed velocities at the various substrate concentrations and the calculated velocities from the MlchaelisMenten equaUon using the best-fit kmetic parameters m&cated that the &stnbution of residuals was r a n d o m [38] and lmphes that the simple Mlchaehs-Menten equation adequately describes the relationship between the velocity and substrate concentration for thrombm and the peptlde pnitroamhde substrates.

Substrate selecttvtty Thrombin cleaves a variety of protems for winch the amino acid sequences around the scissde bond of these proteins differ substantially [8,9] (Fig. 1). Although argtmne ~s most commonly found in the P1 position [39] thrombm will cleave proteins with lyslne m tins position. Prohne or apolar amino acid residues are most frequently found m the P2 positron, i.e., proline in 10 of 18 peptides and lsoleuclne or vahne in four. Although the presence of particular residues in other positrons ~s not as pronounced as for P~ and P2, addmonal subszte amino acid residues undoubtedly contribute to thrombin's specificity for protein substrates. Apolar anuno acids are the most frequent m the P3 positron; however, the variety of amino acids in P3 is great and the frequency of occurrence of any particular type of residue much less than that observed for the P2 positron. It ~s interesting to note that the armno acid sequence at the cleavage site m antlthrombln III is known [40-42], vaz. Ile-Ala-Gly-Arg-. This site is the common locatmn for cleavage by thrombm, Factor Xa and Factor IXa [41], and may serve as an m&cator of a sequence provadmg nummal selectlwty. The second-order rate constant kcat//Km prowdes the simplest single parameter for assessing

the specificity of an enzyme for competing substrates [43]. The tremendous increase m kcat//Km values for the tripeptide substrates (1.07. l0 s M - 1 • s-~ for H-DPhe-Aze-Arg-pNA compared to 6.3. 102 M - 1 . s-1 for Bz-Arg-pNA) suggests a particularly slgmficant role for subslte amino acid residue interactions. Moreover, these values approach those observed w~th enzymes such as catalase and chymotrypsm [43,44]. The preference of thrombm for argtmne over lysme is seen from the observation that kcat//Krn values for the substrates with lysme m the PI position are significantly lower than for those with argmme m tins position. The substitution of lyslne for argmlne m Tos-Gly-Pro-Arg-pNA (Table II), DVal-Leu-Arg-pNA (Table V) and DVal-Phe-Argp N A (Table IV) drops the kcat/K m by 25-, 67- and 95-fold, respectively. These findings are smular to the 10-fold decreased inhibition of thrombm by Phe-AIa-Lys-CH2C1 compared to Phe-Ala-ArgCH2C1 reported by Kettner and Shaw [45]. The sigmficance of prohne in the P2 position is very apparent from the consistent presence of tins anuno acid or ~ts homologs m the substrates w~th the Inghest kcat//Km values. For example, Tos-GlyPro-Lys-pNA ~s the only substrate with lysine m the P1 posmon that has a Ingh kcat/Km and a low K m Substltuuon of prohne (Table II) by phenylalanme (Table V) m DVal-Pro-Arg-pNA drops the kcat//Km by 260-fold, further indicating the contribution of proline in the P2 position. The best substrates investigated have apolar amino acid residues of the D configuraUon in the P3 position. The possible exception to tins is TosGly-Pro-Arg-pNA, winch, however, has no asymmetric center m the P3 anuno acid. The effect of having a 'P4' residue available for interactmn with thrombm is apparent from comparison of BzPyrGlu-Gly-Arg-pNA with PyrGlu-Gly-Arg-pNA (Table V). In tins pair kcat//Km is decreased 100fold in the absence of the 'P4' substltuent. The role of the aromatic residue in the P3 positron can also be seen from the first three entries of Table V, m winch a 10-40-fold decrease is seen for substrates with a relatwely poor aliphatlc apolar anuno acid in the P3 positron rather than an aromatic residue. Valid comparisons between the lanet~c parameters reported m the hterature for pept~de pmtroanilide substrates and those reported here can

554

be made, although not without adjustment for different solution compositions and temperature. If lmtml-velocity estimates are made by methods that do not compensate for significant substrate hydrolysis, any product inhibition will bins esumates of the lmtial velocity and will necessarily bins the values determined for K m with Km(obs) appearing greater than the true value [46]. Such issues have been exarmned by Nlmmo and Atklns [33]. In general, the K m values found in this investigation are lower than those reported by others. Very early data obtained by us prior to implementation of the progress curve fitting for determining imtml velocities produced lugher estimates for K m than those obtained after its implemention. Because the selectivity of the substrate is due to the peptlde, compeutive product inhibition Is expected and has been observed with several substrates (Lottenberg et al., unpubhshed observations) The lonetic parameters for actwe-s~te tltrated bovane thrombin for Bz-Arg-pNA determined by Takasakl et al. [47] are in good agreement with our data, although their K m is 2-fold higher Mlchaehs constants for bovine thrombln acting on Bz-PheVal-Arg-pNA and Bz-(NO2)Phe-Val-Arg-pNA have been reported [48] with the K m values m that study dxffenng from those reported here but not in a systematic way. The Mlchaehs constants for human a-thrombm reported in that study are very similar to those observed here; however, their kca t values are approximately one half. KmeUc parameters for bovine and human a-thrombm hydrolysis of Tos-Gly-Pro-Arg-pNA and oPhe-Plp-Arg-pNA have K m values that are the same wltlun error [28] The kc, t values are different, with kcat for Tos-GlyPro-Arg-pNA being 20% higher with human thrombm and that with oPhe-Plp-Arg-pNA being 10% lower [28] than for the bowne enzymes. Pozsgay et al. [49] have examined the hydrolysis of 36 pept~de p-mtroamlide substrates by human thrombin at 37°C, five of which are the same as those mvesUgated here with bovine thrombxn at 25°C. Differences of 2- to 5-fold in both K m and k~at are observed even after 'correction' of the data to comparable con&tions of temperature and ionic strength. Data obtained by Christensen and coworkers for human thrombln [50] at both 25 and 37°C agree very well with those of this study, but

with K m values that are higher. Similar &fferences exist for DPhe-Plp-Arg-pNA and Bz-Phe-Val-Argp N A reported In the manufacturer's literature (see Ref 50); Le., K m values are higher for both bovine and human thrombins, but where available k~t values are in good agreement. Collen et al. [51] found very high K m values and low kc, t values for substrates with lysme in the PI posmon [51], consistent with our data for s~rmlar substrates. McRae et al. [52] have reported lonetlc parameters for thrombm and peptide thloester substrates No ldenUcal peptides were employed m that study and this one, but several of the thioesters are sufficiently close in structure to make general comparisons possible. For example, very slrmlar values for kcat/Km, kca t and K m for Cbz-Gly-Arg-SBu and Bz-PyrGlu-Gly-Arg-pNA (Table III) and BocVal-Phe-Arg-SBz and H-DVal-Phe-Arg-pNA are observed, but m the latter case K m is tugher by about 3-fold. Several possible mechanisms for the effect of poly(ethylene glycol) on the K m of thrombin for both of the substrates studied may be considered. D~rect interaction between the substrates and the polymer, or polymer-related alteration in the solubdlty or state of association of the substrate would both give nse to an apparent increase in Km. Aggregation m aqueous solution has been reported for Pro-Leu-Gly analde [53] and poly(ethylene glycol) can dramatically promote self-associaUon of some proteins [31] The observation of an effect on K m for Bz-Arg-pNA, where K m may be approximately equal to K s, the enzyme-substrate dlsSoclat'.on constant, argues that the simplest explanauon for the effect is reduction in solubility of the substrate. An addmonal posslblhty to consider for Tos-Gly-Pro-Lys-pNA is that alteraUon m bulk solution viscosity increases the apparent K m as a result of its reducing kl, the bimolecular rate constant for enzyme-substrate association. Renard and Fersht [44] have argued that the bimolecular step in chymotrypsin hydrolysis of substrates for which kcat//gm IS 5.6 107 M - ' s-J may be rate llnuting Evidence to support this explanation was found from the linear dependence of kcat//Km o n the reciprocal of the viscosity of the poly(ethylene glycol) solution as predicted for a dlffUSlOn-hrmted reaction [54]. The viscosity data were for PEG 4000 (prowded through the courtesy of Kenneth

555 Ingham) rather than 6000, as no data for PEG 6000 were found in the literature. The increases observed i n K m with increasing NaC1 may also be related to a decrease in the concentration of monomerlc substrate, slrmlar to one explanation for the effects of poly(ethylene glycol), although no direct investigation of this has been made. The lsoelectric p H for thrombln is reported to be approx. 7 5 (Fass, D. and Mann, K.G., personal communication, and Refs 55 and 56), and thus electrostatic effects of a magnitude to alter K m by 5-fold appear unlikely Data from investigation of the kinetic parameters for hydrolysis of Tos-Gly-Pro-Arg-pNA by bovine fl-thrombin (Lottenberg et al., unpubhshed observations) and the effect of NaC1 concentration on i t s K m and kcat suggests a direct effect of NaC1 on athrombm. Whereas K m and kca t for a-thrombm increase from 3.6 to 8.4 pM and 102 to 124 s-i, respectively, m going from 0.1 to 1.0 M NaC1, both K~ and kca t a r e the same for fl-thrombin, namely 14 /tM and 124 s -~ at both NaC1 concentrations. Effects of ionic strength and lomc composmon on the thermal stabihty of human thrombm can be interpreted as indicating that sigmficant structure alteration occurs with change m electrolyte concentration and composition, and thus the changes that are observed as perturbing the thermal stablhty also may perturb the structure m such a way that a-thrombm becomes more like fl-thrombln [58]. Ca 2÷ effects on thrombin hydrolysis of TosGly-Pro-Arg-pNA are very small at constant ionic strength of 0 107; however, at high ionic strength Ca 2÷ is seen to markedly i n c r e a s e Kin, approxamately 3-fold compared with NaC1 at the same calculated ionic strength. In contrast to NaC1, CaC12 decreases kca t t o 73 s-J from 102 s - l at ionic strength 0.107 M, and to 73 s -~ from 108 s-1 at iomc strength 0.5 M Until ionic strength 0.5 is reached, or no added NaC1 is present, there IS no sigmflcant effect o n g m . In view of the reported effects of Na ÷ on thrombin [58,59], tins latter observation cannot be uniquely interpreted. The pH dependence of keat/Km for the hydrolysis of Tos-Gly-Pro-Arg-pNA and Tos-Gly-ProLys-pNA by thrombln (Fig. 3) indicates slgmficant difference in the behavior of thrombln activity with p H from that usually observed with the

pancreatic serlne proteinases on low molecular weight substrates. Recent pre-steady-state data for bovine thrombm hydrolysis of Z-Lys mtrophenyl ester show a snmlar inflection [60]. Inflections are also seen in the graph of kea t vs. pH, indicating complex pH dependence that prevents interpretation on the basis of p K values of the functional groups participating in the hydrolytic reaction. From the armno acid sequence data, VlZ the presence of residues equivalent to His-57, Asp-102, Asp-185 and Ser-195 of chymotrypsm [61] and the slrmlarlty in reactlvaty to dilsopropylphosphorofluondate and peptlde chloromethyketones [45], the basic mechanism of hydrolysis is concluded to be slrmlar to that of trypsin and chymotrypsln. Consequently, the p H dependence of the lonetic parameters (Tables Villa and VIIIb) serve to indicate possible active-site &fferences that may account for the Ingh selectivity of thrombln. Thts is further indicated by the general observations that may be drawn from plots of log kca t and l o g K m versus pH. For Km, the slope of the lines that intersect the line of zero slope at the maxamum have a slope of - 1 at the low-pH side of the data, whereas for kca t the slopes are either - 2 or 3 for the two substrates, suggestwe of more than one proton transfer being involved in the catalytic process [30]. In cases of such complexity, the advice of Knowles [62] on interpretation of pH dependence of steady-state kinetic parameters appears particularly relevant. When sufficiently extensive laneUc data of adequate accuracy become available for the other coagulation proteinases, accurate deterrmnation of concentrations of individual coagulation proteinases, even m rmxtures, becomes possible m principle, and, depending on the actual selectiwty of the m&vldual peptide substrates for particular proteinases, entirely practical. The very large range in k c a t / K m values for the various peptlde p-nltroanlhdes investigated here in&cates considerable potential for development of Inghly selective assays for thrombin, even in the presence of other protelnases. Prelirmnary evidence (Jackson and Hall, unpublished data) indicate that thrombin and Factor Xa can be readily assayed m the presence of each other with an accuracy better than _+5% when DPhe-Pip-Arg-pNA and Bz-IleGlu-Gly-Arg-pNA are used as preferential sub-

556

strates for thrombm and Factor Xa, respectwely. Moreover, current availabihty of mexpenswe computers makes it possible to envision routine accurate measurement of specific plasma protemases from systems in addmon to the coagulation system, m the presence of other proteolyt~c enzymes of smular but distinguishable specificity, m both chmcal enwronments and in the research laboratory.

Acknowledgements The authors would like to thank Kathenne K. Lawne, Wllham Salsgtver and Ingemar Bjork for the preparation of the thrombm, K.K. Lavlne for perfornung the bioassays, Kenneth Ingham for providing viscosity data for PEG 4000, Debra Hodak for her assistance xn preparing the manuscript, and Drs. Thomas Carhsle and Luis Glaser for commenting on the manuscript. Tlus work was supported by grants from the U.S. National Institutes of Health, HL 12820 and a trammg grant HL 7088.

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