Eur. J. Biochem. 86, 353-360 (1978)

Kinetics of the Interaction of a-Chymotrypsin with Trypsin Kallikrein Inhibitor (Kunitz) in Which the Reactive-Site Peptide Bond Lys-15 - Ala-16 is Split Ulrich QUAST, Jurgen ENGEL, Erna STEFFEN, Harald TSCHESCHE, and Sigrid KUPFER Abteilung fur Biophysikalische Chemie. Biozentrum der Universitit Basel, and Abteilung fur Organischc Chemie und Biochemie, Technische UniversitHt Miinchen (Received November 24, 1977)

Modified trypsin kallikrein inhibitor (I*), with the reactive-site peptide bond Lys-15 - Ala-16 split, reacts with a-chymotrypsin (E) via an intermediate X to the stable tetrahedral complex C : E + I*.+ X + C. Formation of X constitutes a fast pre-equilibrium (equilibrium constant K, = 7 x l o p 5M, association rate constant k , = 4 x lo3 M - ' s - ') to the slow reaction X + C (rate constant k , = 2 x s-'), all values at pH 7.5. No intermediate X is observed when a-chymotrypsin reacts with I*OMe in which the carboxyl group of Lys-15 is esterified by methanol. This observation as well as the different pH dependence of the overall association rate constants in the case of I* and I*-OMe indicate that formation of X precedes formation of the acyl enzyme in the catalytic pathway. The data are compared to the similar results obtained with P-trypsin and I* or I*-OMe.

Trypsin kallikrein inhibitor (I), like many protein proteinase inhibitors [I], binds tightly to specific proteinases in a substrate-like way [2]. The binding site of the inhibitor contains a reactive-site peptide bond, i. e. Lys-15-Ala-16 in the case of I. This bond is catalytically split by the enzyme. The reaction in which modified inhibitor I* is formed leads to an equilibrium [I*]/[I] = Khrdnear unity for many inhibitors [3] at neutral pH. In the case of trypsin kallikrein inhibitor the slow cleavage of the reactive site peptide 'bond remained first undetected [4] but recently Tschesche and Kupfer found equilibrium constants Khrd= 0.3 0.38 at pH 5-7.5 [5,6]. The time needed to reach equilibrium was one year. The enzymatic hydrolysis was confirmed by Estell et al. [7], who showed that incubation of virgin inhibitor with a protease from starfish at pH 10 leads to almost complete conversion from I to I*. In a previous communication [8] we have shown that the reaction of chymotrypsin with both virgin inhibitor I and modified inhibitor I* leads to the same complex C. From detailed X-ray crystallographic studies [9 - 111it is known that in C the carboxyl group of .-

Ahhreviutions. I, virgin trypsin kallikrein inhibitor (Kunitz) from bovine pancreas; I*, modified inhibitor in which the reactive site peptide bond Lys-15-Ala-16 is open; I*-OMe, I* with the Lys-15 carboxql group and probably all other frcc carboxyl groups esterified by methyl alcohol; [ lo denotes total concentration. Enzymes. oc-Chymotrypsin (EC 3.4.21 , I ) ; fl-trypsin (EC 3.4.21.4).

Lys-15 is in a tetrahedral state with the reactive-site peptide bond intact. In the reaction with virgin inhibitor, Cis formed via a Michaelis complex L, which is in fast pre-equilibrium with inhibitor and enzyme with an equilibrium constant KL = 5 x M [12]. From I* formation of C is about 2 x lo4 times slower than from I [8]. According to the catalytic mechanism of the serine histidine proteases [2,13], several intermediates can be expected in this reaction, which leads from the open peptide bond in I* to the tetrahedral complex C. Here we present evidence for an intermediate in the reaction of I* with a-chymotrypsin, the formation of which is much slower than expected for the Michaelis complex.

MATERIALS AND METHODS Virgin trypsin kallikrein inhibitor was a kind gift of Bayer AG (Wuppertal, F.R.G.). It was characterized as described before [12,14]. The molar absorption coefficient was E~~~ = 5.4 x l o 3 M - cm-'. The inhibitor was converted to I* according to Jering and Tschesche [15,16] and I*-OMe was prepared by esterification of I* with methanol [14]. Both materials were purified and characterized as described earlier [14]. Absorption coefficients = 6.1 x lo3 M - ' cm-I for I* and 7 x lo3 M- ' cm-' for I*-OMe [14]. a-Chymotrypsin (3 x crystallized, lot CDS 54C404 from Worthington) was checked for its purity by dodecylsulfate gel electrophoresis. Only a 5 impurity of molecular weight

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Interaction of Chymotrypsin with Modified Inhibitor (Kunitz) 1

I

1

1

I

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--

-40 000 was detected which was enzymatically inactive. - 0.5 The molar absorption coefficient of the enzyme = 5 .ix 1 0 4 M - 1 cm-' was determined by active-site - 0.2 titration with 4-nitrophenyl-N'-acetyI-N1-ben0zylcarbazate according to Elmore and Smyth [17]. 2 zo.1 Proflavine was purchased from Fluka (Fuchs, -b T Switzerland) and 4-nitrophenyl-NL-acetyl-N1-benzyl-- 0.05 carbazate from Nutritional Biochemicals Corp. (Cleveland, Ohio). All other chemicals were reagent grade - 0.02 (Merck, Darmstadt). All protein concentrations were based on molar absorption coefficients and all mea0 8 16 20 " 48 88 128 surements were performed at 22.5 "C. T i m e (s) Stopped-flow experiments were carried out with a Fig. I . Fast phuse of the association ofcc-chymotrypsin nith modified Durrum Gibson stopped-flow photometer which has inhibitor. The concentrations in the stopped-flow cell were [enzyme], = 2.9 pM, [I*], = 23.3 pM, and [proflavine], = 30 pM in0.05 M Tris been greatly improved by Dr G . Hanisch from our buffer, pH 7.5, containing 0.2 M KC1 and 0.05 M CaCI,. The change department [14]. The data were stored with a transient of absorbance 6 A due to the displacement of proflavine by the recorder (Datalab DL 905) which was connected on line inhibitor was recorded at 465 nm. The reaction starts at an amplitude to a PDP 11/40 computer. The proflavine displacement 6A,,, and levels off at a value (---) which was set to zero. At the contracted time scale the beginning of a slow phase (see Fig. 2) is method [12] was used to follow the association of c(observed. From a semilogarithmic plot of 6A/6AI 0 versus time chymotrypsin with inhibitor. The absorbance change ( 0 4 )a pseudo-first-order rate constant of k,,, = 0.35 s-l was due to the displacement of proflavine by the inhibitor is obtained after correction for the presence of proflavine proportional to the change of free enzyme concentration [12] and was monitored at 465nm. All association rate constants as well as equilibrium constants k 41.0 which were determined by this method were corrected h\ for the fraction of enzyme occupied by proflavine [12]. 2.5 About eight measurements of each concentration were I \ I \ averaged and analyzed as described under Results. The slow phase of the association of I* with chymotrypsin was followed by proflavine displacement in a thermostatted Shimadzu UV 200 double-beam 0.05 spectrophotometer with an appropriate chymotrypsin/proflavine solution in the reference cell. u \ 0.5 - 0.02 Free chymotrypsin was determined by enzymatic test with N-(3-carboxypropionyl)-phenylalanine-4"0 2 4 6 8 10 12 nitroanilide as the substrate and monitored at 405 nm Time (ks) [18]. The reaction was monitored at 405nm and the Fig. 2. Slow phase qf the association oj'cc-chymotrypsin with modified concentration of free enzyme was determined from inhihitor. The reaction was followed by the change of absorbance 6 A at 465 nm after mixing the reaction partners in a spectrophotometer initial rates.

.

=

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\

RESULTS Evidence j o r an Intermediate

The association of modified inhibitor I* with uchymotrypsin occurs in two distinct kinetic phases. The fast phase was resolved in a stopped-flow photometer (Fig. 1). The slow phase, which at the given concentration has a much larger amplitude, can be conveniently observed after conventional mixing in a spectrophotometer cuvette (Fig. 2). This phase has already been described in an earlier publication [8].The pseudo-first-order rate constants of the phases as well as their amplitudes were obtained from experiments under pseudo-first-order conditions [I*], + [enzyme], like those shown in Fig. 1 and 2. For the experimental

cuvette. The fast phase (Fig. 1) was already over within the dead-time mixing. 6 A , = is the amplitude at zero time and the final value was set to tero. The concentrations after mixing were [enzyme], = 3.36 pM. [I*], = 35 pM and [proflavine], = 22.4 pM in 0.1 M sodium phosphate buffer, p H 7. From a semilogarithmic plot of 6A/6AI=, versus time a pieudo-first-order rate constant of /can" = 6.1 x s - was obtained (corrected for proflavine)

conditions of Fig. 1 and 2 the following values were obtained: kspp= 0.35 s-' for the fast phase and kapp = 6.1 ~ 1 0sC1 for the slow phase. The normalized amplitude of the fast phase 6 A x / 6 A , was 0.16, where h A , = 6 A x + 6A,,,, is the total amplitude. In order to decide whether the fast phase reflected the formation of an intermediate X or whether it was caused by a faster reacting impurity in the I* preparation, a solution of 3 pM chymotrypsin was reacted with

U. Quast, J. Engel, E. Steffen, H. Tschesche, and S. Kupfer I

I

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time-scale on which C is formed, X is in equilibrium with the free reactants:

I,..

0.15

From the concentration dependence of the normalized amplitude of the fast process 6Ax/6Ao,one can calculate K,, since bA,/6Ao = [X]/[E],. Fig. 3 shows that a good fit was obtained with K, = 6.5 x M. With this equilibrium constant and with the value of the apparent rate constant of the fast phase kapp= 0.35 s - ' (see Fig. 1) the rate constant k , in mechanism (1) was calculated according to