A fluorescent study of urokinase using active-site directed probes

Biochimica et Biophysica Acta, 704 (1982) 403-413 Elsevier Biomedical Press

403

BBA31181

A FLUORESCENT STUDY OF UROKINASE USING ACTIVE-SITE DIRECTED PROBES GUENTHER SCHOELLMANN a.,, GEORGE STRIKER b and ENG BEE ONG c

a Department of Biochemistry, Tulane University Medical Center, New Orleans, LA 70112 (U.S.A.), b Max-Planck.Institute of Biophysical Chemistry, 34 G6ttingen (F.R.G.) and c Department of Pathology, New York University Medical Center, New York, N Y 10016 (U.S.A.) (Received January 21 st, 1982)

Key words: Fluorescent probe," Urokinase

Human high and low molecular weight forms of urokinase (EC 3.4.21.31) were eovalently labeled with two active-site directed fluorescent probes, dansyi fluoride and Dns-Gly-Nle-Lys-CH2CI. Highly purified samples of both derivatives were obtained using affinity chromatography with Sepharose 4B-e-aminocaproyl-paminobenzamidine and Sepharose 4B-c-aminocaproylagmatine columns. The peptide chloroketone selectively reacted with an active-site histidine residue, whereas the dansyl-fluoride was covalently attached to the active-site serine residue. Fluorescence lifetimes of the probe-modified urokinase samples were measured in water and deuterium oxide with a photo-counting technique and the decay data were analyzed by a method developed by Striker (unpublished data). Spectral properties of the urokinase derivatives were compared with model compounds investigated previously (Vaz, W.L.C. and Schoeilmann, G. (1976) Biochim. Biophys. Acta 439, 206-218) and with the new model compound Dns-Gly-Nle-Lys-CH2CI in various solvents of different polarity. The fluorescent emission transition energies were correlated with the solvent polarity parameter ' Z ' of Kosower (Kosower, E.M. (1968) An Introduction to Physical Organic Chemistry, ch. 2.6, p. 293, J. Wiley, New York). The enhancement effect of deuterium oxide on several fluorescent parameters was used to estimate solvent accessibilities to the chromophore bound to the enzymes. With an estimated Z value of 57, the primary binding sites of high and low molecular weight urokinase were found to be considerably more apolar than the corresponding site in trypsin. On the other hand, the solvent accessibility of 65-70% is somewhat greater than for trypsin. The absence of significant differences between the different forms of urokinase indicates identical catalytic sites. A binding subsite, probably S4, of relatively high polarity ( Z --87.6) has been probed by the fluorescent peptide chloroketone; however, the 20% solvent accessibility to this pocket is very low. We postulate the existence of a specific binding pocket in the general area around subsite S4 in urokinase which might have a decisive function in the selectivity and high specificity of the plasminogen activators. Small differences in solvent accessibility were found for high and low molecular weight forms of urokinase and support reported differences in relative enzymic activity between the two forms (Wohi, R.C., Summaria, L. and Robbins, K.L. (1980) J. Biol. Chem. 255, 2005).

Introduction Several cellular and tissue plasminogen activators are involved in pathological transformations, * To whom correspondence should be addressed. Abbreviations: Dns, dansyl; Nle, norleucyl; DGNL-CM, DnsGly-Nle-Lys-CH2C1. 0167-4838/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

including oncogenesis [1], inflammation [2], cocarcinogenesis [3], and in a number of thromboembolic disorders. Urokinase (EC 3.4.21.31), a proteolyltic enzyme with trypsin-like specificity for synthetic and natural substrates, is known to be a plasminogen activator. Two forms of human urokinase with different molecular weights have

404 been characterized by affinity chromatography on Sepharose-c-aminocaproylagmatine [4] or Sepharose-c-aminocaproyl-p-aminobenzamidine [5]. A standard approach to obtain topographical information about binding sites of macromolecules is to develop a series of specific substrates and inhibitors and then study the kinetics of their interaction with the macromolecule. Very useful information results from these methods, as recently shown with urokinase [6-8]. But investigation and comparison of closely related enzymes, e.g., those from the fibrinolytic system, reveal poor resolution and often cross-reactivity. An approach is needed which gauges 'topographic differences and features of primary nature', in other words, methods, in which the inherent chemical reactivity of substrates and inhibitors (such as the sequencerelated chemical reactivity of an ester bond hydrolyzed during action of an enzyme on the substrate) does not play a role in the deductive process. Active-site directed, irreversible labeling with simultaneous incorporation of a chemo-optical probe appears to be capable of fulfilling such requirements. Fluorescent probes are potentially well-suited because of the many parameters that can be correlated with structural features of the immediate microenvironment of the dye. In addition, longrange energy-transfer measurements can provide information on even remote parts of the macromolecule. This approach, originally suggested and used independently by Haugland and Stryer [9] and Schoellmann [10], has been applied to several enzyme systems including plasma proteins [ 11-15]. Utilizing these ideas, we have investigated fluorescence probe-modified urokinase. Dansyl fluoride, previously shown to sulfonate the active-site serine residue of other proteases in a highly selective manner [12,13], was used to monitor the primary catalytic site, whereas the peptide chloroketone Dns-Gly-NIe-Lys-CH2C1 , which alkylates the active-site histidine residue, was expected to probe some parts of the extended binding sites of this enzyme.

Experimental methods

Preparation of Dns-Gly-Nle. Gly-Nle (188 mg, 1 mmol) was suspended in 25 ml ethanol. Freshly

distilled triethylamine was added in an equivalent quantity, followed by the addition of 0.2 ml of a 5% aqueous sodium bicarbonate solution. To the suspension was added dansyl chloride (378 mg, 1.4 mmol) dissolved in 10 ml ethanol. The pH of the solution was adjusted with triethylamine to 9.0 and the mixture stirred for 5 h in the dark at room temperature. After 2 h the pH of the solution had dropped to 8.2 and was readjusted to 9.0 with more triethylamine. The solvent was finally removed under vacuum and the residue dried overnight. The crude product was purified by passing it through a silica gel column (1.5 × 35 cm) using Bio-Sil A, 200-325 mesh (a product of Bio-Rad). An analytically pure sample emerged between 60180 ml with ethyl acetate/methanol mixtures of increasing polarity as developing solvents. After evaporation of the solvent 218 mg of Dns-Gly-Nle (55% yield) were obtained. Analysis for C20H27N3OsS: Calc.: C = 56.99%; H = 6.46%; N = 9.97% Found: C = 56.90%; H = 6.40%; N = 9.45%;

Preparation of Boc-Lys(Z)-CH2CI. This compound was prepared from Boc-Lys(Z)-OH (1.5 g, 3.95 mmol) by activation with freshly distilled ethyl chloroformate (0.42 ml, 4.35 mmol) at -15°C in anhydrous tetrahydrofuran followed by reaction with etheral diazomethane solution. The mixture was kept at 0°C for 30 min with stirring. After washing the etheral solution with 0.1 M acetic acid and saturated sodium bicarbonate solution, the resulting diazomethyl ketone was treated with cold 5 M HC1/ethanol until nitrogen evolution had ceased (approx. 2 min). In order not to deblock the N-terminal protective group at this stage, the acid treatment must be kept to a minimum. The resulting solution was washed once with cold 1 M HC1 and once with saturated sodium bicarbonate solution. Solvent was removed and 1.39 g (86% yield) of a white solid obtained (m.p. 83-85°C). Preparation of Dns-GIy-NIe-Lys(Z)-CH2CI. BocLys(Z)-CH2CI (500 mg, 1.31 mmol) dissolved in 3 ml dry ethanol was deblocked at the a-amino group with 2 ml 5 M HC1/ethanol (10 mmol) by stirring the mixture for 60 rain at room temperature. After evaporation of the solvent and triturating the white, solid mass with ether, the hydrochlo-

405 ride was obtained and was used without further purification. Dns-Gly-Nle (134 mg, 0.32 mmol) dissolved in 4 ml dry tetrahydrofuran was activated with ethyl chloroformate and equimolar amounts of triethylamine at - 1 5 ° C for exactly 9min. This mixed anhydride preparation was then combined with a precooled solution of NH2-Lys(Z)-CH2C1.HC1 (112 mg, 0.31 mmol) in tetrahydrofuran containing enough triethylamine to neutralize the hydrochloride. After the temperature was raised slowly to 0°C, the reaction mixture was stirred for 30 min, followed by 45 min at room temperature. The mixture was taken up in 40 ml ether, washed with water, 0.1 M HC1 and saturated sodium bicarbonate solution. After removal of the solvent the desired compound was obtained (170 mg, 70% yield) is a light-tan amorphous solid which showed a single spot on TLC (silica gel G, Merck A.G.) using methyl ethyl ketone/pyridine/water/acetic acid (70: 15 : 15 : 2) as developing solvent.

Preparation of Dns-GIy-NIe-Lys-CH2CI. HCI. Dns-GIy-Nle-Lys(Z)-CH2CI (170 mg, 0.23 mmol) was heated in 2 ml trifluoroacetic acid for 1 h at 50°C. After cooling in an ice-bath 12 ml 3.5 M HC1/ether was added. After standing for 30 rain the white precipitate of the hydrochloride salt was collected and washed with minimal amounts of dry ether and yielded 120 mg (86%) of the desired product. Analysis for C27H41C12NsOsS: Calc.: C = 52.42%; H = 6.68%; N -- 11.32% Found: C=52.04%; H=6.54%; N=11.18%.

Dansyl fluoride. This compound was prepared as described previously [12]. Purification of urokinase. The high molecular weight form of urokinase (the high and low molecular weight forms of our preparations found by SDS-acrylamide gel electrophoresis to be 33400 and 51700 daltons will be designated here 33 kDa urokinase and 52 kDa urokinase) arid the low molecular weight form were isolated and purified from partially purified human urokinase (Serano, Lot 207 with a spec. act. of 60400 I U / m g protein and Sterling Winthrop, Lot R005JF with 40000 I U / m g protein). Each sample was pre-purified by the method of Holmberg et al. [5] with the use of

Sepharose 4B-c-aminocaproyl-p-aminobenzamidine. Final purification was achieved with the use of Sepharose-c-aminocaproylagmatine [4]. A sample of 52 kDa urokinase (Sterling Winthrop) was purified and autocatalytically converted to the low molecular weight urokinase [16]. After incubation in the presence of 0.04% sodium azide at 37°C for 25 days, the fully converted material was passed through a Sepharose 4B-caminocaproyl-p-aminobenzamidine column to remove the cleaved 18 kDa fragment. The specific activities of 52 kDa urokinase and 33 kDa urokinase were 122000 and 185000 I U / m g protein, respectively; the respective molar activities were 1.00.1013 and 1.15- 1013 IU/mol activesite titrant. All samples appeared to be homogeneous as determined by SDS-acrylamide gel electrophoresis by a modification of the method of Weber and Osborn [17]. 52 kDa urokinase also revealed trace amounts of 33 kDa urokinase ( < 5%). The enzymic activity was estimated by the spectrophotometric assay with pyroglutamylglycylarginine p-nitroanilide (S-2444) [18]. The protein concentration was determined by ultraviolet absorption at 280 nm using E ~ m= 12.6 [4] and active-site titration was carried out with pnitrophenyl-p'-guanidinobenzoate [ 19].

Preparation of Dns-Gly-Nle-Lys (DGNL)-derivatives of each form of urokinase. DGNL-33 kDa urokinase and DGNL-52 kDa urokinase were prepared by reacting each form of the urokinase with DGNL-CM [20]. To an ice-chilled solution 0.77 mM 33 kDa urokinase of 52 kDa urokinase (Sterling Winthrop) in 0.2 M NaCI and 0.12 M sodium phosphate buffer, pH 7.5, was added an equal volume of DGNL-CM (4.33 mM) dissolved in 1 mM HCI. The reaction mixtures were incubated at 37°C for 1 h. The inactivated samples were exhaustively dialyzed for at least 24h against several changes of water at 4°C. They were freeze-dried and kept under refrigeration below 0°C until needed. The reaction sample and all fluorescent derivatives were handled with great care to minimize exposure to light. The inhibition was followed by assaying aliquots of samples withdrawn from the incubation mixture at various time intervals. Proof of homogeneity of the fluorescent derivatives and stoichiometry of incorporation of the dansyl peptide into each form of

406

urokinase were obtained by SDS-acrylamide gel electrophoresis and quantitative amino acid analysis [21] before and after performic acid oxidation [22].

Preparation and purification of the dansyl fluoride derivative of each form of urokinase. Each form of urokinase, 33 kDa urokinase (Serano), 33 kDa urokinase C and 52 kDa urokinase (Sterling Winthrop), was reacted with dansyl fluoride essentially according to the method of Vaz and Schoellmann [12]. Each reaction mixture was incubated at 37°C in 6% isopropanol and then terminated after 5 h. Since the urokinase was not completely inactivated, the dansyl derivatives were isolated from the residual active enzyme by passing the samples immediately through Sepharose 4B-~-aminocaproyl-p-aminobenzamidine columns, followed by exhaustive dialysis overnight against several changes of 0.05 M ammonium bicarbonate solution at 4°C. Solutions of the fluorescent derivatives were lyophilized, sealed and stored below 0°C. Fluorescence measurements. Corrected fluorescence spectra were obtained at 20°C on a FICA 55 Absolute Spectrofluorimeter with excitation bandwidth set at 7.5 nm and emission bandwidth set at 2.5 nm. Scanning speed was 10 nm/min. Lyophilized protein samples were dissolved in potassium phosphate buffer, 0.1 M, pH 7.0 (p2H 7.0) prepared either with water or deuterium oxide. All

m

'~

20 NS

-

solutions were adjusted to an absorbancy of 0.1 before taking the spectra. Time-resolved fluorescence spectra were obtained by exciting the solutions with light pulses and measuring the time course of fluorescence decay by the single photon counting technique. An ORTEC 9250 Nanosecond Fluorescence Spectrometer with an ORTEC Nanosecond Light Pulser Model 9352 was used. Correction for light pulse and deconvolution analysis was done with a new method developed by Striker (unpublished data) at the Max-Planck-Institute for Biophysical Chemistry, G6ttingen, F.R.G. A characteristic plot is shown in Fig. 1. A few samples were excited with an N2-1aser (full pulse width 4 ns) and an analysis will be described in detail elsewhere. No deconvolution analysis was done for these samples. Results

Synthesis of DGNL-CM Previous studies have demonstrated that peptide chloroketones with a minimum length of three residues and a C-terminal basic amino acid residue are specific irreversible inhibitors for both molecular forms of urokinase [6,23]. Utilizing these findings, we have synthesized a tripeptide chloroketone dansylated at the N-terminal end [20]. Choice of the amino acid residues was dictated by the desire to incorporate into the proteins an atypical amino acid residue easily identifiable by analysis. The dansyl group, on the other hand, would permit the probing of extended active-site structures of the various forms of urokinase. The peptide chloroketone synthesized using the mixed anhydride method was obtained in good yield. The compound was tested as an affinity label for trypsin-like enzymes by reaction with trypsin. Specific incorporation of the peptide moiety and exclusive reaction with the active-site histidine residue (His-46) of trypsin was verified.

Dansyl fluoride 104

208

312 416 Chonnel

520

624

728

832

Fig. 1. Time-resolved fluorescence decay of 33 kDa dansyl fluoride-urokinase in deuterium oxide at 20°C. Solid line through the curve: best-fit curve for a species with two decaying parameters, 3.4 and 19.9 ns. Peak=9.77.103 at channel 82; F-PK (peak exciting light pulse)=2.78, l04 at channel 56; analysis starts at channel 50; 0.11689 ns/channel.

Dansyl fluoride was employed as an active-site directed fluorescent probe which should react covalently in the primary binding site of urokinase or in close proximity to such a site. We have previously shown that dansyl fluoride is a highly specific fluorescent probe for serine proteases [12], results confirmed by Berliner and Shen [13].

407

Fluorescent derivatives of high and low molecular weight forms of urokinase The peptide chloroketone Dns-Gly-Nle-LysCH2C1 is only a slightly less effective inhibitor than the earlier reported chloroketone with the sequence Nle-Gly-Lys-CH2C1 [23]. This is probably due to a little less favorable interaction with the S2-subsite (nomenclature of Schechter and Berger [26]) of the enzyme. Other investigators [27-29] reported similar findings at the P2 position of synthetic substrates with decreased affinity. Nevertheless, the enzyme preparations were rapidly and conveniently inactivated within 15 min, and highly purified preparations of fluorescent urokinase derivatives were obtained. Inactivation of urokinase had to be completed in the shortest possible period to prevent autocatalytic conversion of the high molecular weight form to the low molecular weight form. On SDS-acrylamide gel electrophoresis unreduced and reduced samples of DGNL-33 kDa urokinase each showed a single band stained with Coomassie brilliant blue (Fig. 2A, c and d, left panel). The unreduced DGNL-52 kDa urokinase preparation revealed less than 5% of the DGNL-33 kDa urokinase derivative. The reduced sample A

B

8R33-

33-

10-

a

bc

d

abc

d

a

b

c

Fig. 2. (A) SDS-acrylamide gel electrophoresis on 7.5% gel of Dns-Gly-Nle-Lys-CH2Cl-treated urokinase. The left panel shows a photograph of the gel stained with Coomassie brilliant blue, while the right panel shows one taken under ultraviolet light exposure prior to staining. (a) DGNL-52 kDa urokinase (Sterling), reduced; (b) DGNL-52 kDa urokinase (Sterling), unreduced; (c) DGNL-33 kDa urokinase (Sterling), reduced; (d) DGNL-33 kDa urokinase (Sterling), unreduced. The numbers on the left in descending order denote 52 kDa urokinase, 33 kDa urokinase or heavy chain of 52 kDa urokinase, and the light chain of 52 kDa urokinase. (B) SDS-acrylamide gel electrophoresis on 7.5% gel of unreduced dansyl-fluoride treated urokinase. (a) DF-55 kDa urokinase (Sterling); (b) DF-33 kDa urokinase C; (c) DF-33 kDa urokinase (Serano). Left-hand panel, Coomassie staining; fight-hand panel, ultraviolet light exposure.

showed two stained bands (33100 and 18600 daltons) but fluorescence was noted only in the 33 kDa band, as shown in Fig. 2A, a and b. As expected, modification took place at the histidine residue, as confirmed by the recovery of norleucine as marker and 3-carboxymethyl histidine and a decrease in histidine content after acid hydrolysis before and after performic acid oxidation of the samples. These findings agree with those reported for Nle-Gly-Lys-urokinase, indicating that alkylation with the fluorescent peptide chloroketone occurs at the active-site histidine residue which is in the heavy chain [23]. We found no noticeable difference in the rate of inhibition of the low and high molecular weight forms, but made no detailed kinetic investigation.

Dansyl fluoride-urokinase The reaction of dansyl fluoride with either form of urokinase did not go to completion and was in general much slower than inactivation with peptide chloroketones. The reaction was highly specific, however, and a loss in activity followed incorporation of the dansyl group in a fashion similar to that described for other serine proteases [12,13]. Because of the low solubility of dansyl fluoride in aqueous solutions the probe had to be dissolved in organic solvents. But even then it was impossible to reach high enough concentrations to force a more rapid reaction. Since stability of urokinase preparations is also critical, the reaction time has been limited to 5 h and the final concentration of organic solvent (isopropanol) to 6%. Under such conditions 68-74% of the enzyme preparation could be inactivated. Each of the three dansyl fluoride-treated urokinase preparations were further purified by Sepharose 4B-c-aminocaproyl-paminobenzamidine chromatography. This step was necessary to minimize autocatalytic conversion of the high molecular weight form and to achieve optimal homogeneous fluorescent preparations. The fluorescent derivatives eluted with the initial condition buffer showed negligible activity ranging from 1-4% of initial enzyme activity. The amount of protein in these samples represents 68-75% of the initial enzyme and these values compared favorable with the degree of inactivation of the enzyme preparations (68-74%). These data support the suggestion that the reaction between

408

dansyl fluoride and urokinase is specific. As shown in Fig. 2B, SDS-acrylamide gel electrophoresis of both low molecular weight urokinase preparations (dansyl fluoride-33 kDa urokinase and dansyl fluoride-33 kDa urokinase C) revealed a single band with Coomassie brilliant blue (left panel, b and c) corresponding to a single fluorescent band prior to staining (right panel). The high molecular weight urokinase preparation contained less than 10% of low molecular weight urokinase derivative.

tion could in turn give some insight into the structural relationship of the high and low molecular weight forms of urokinase. Incorporation of the dansyl group into the converted form proceeded similarily and levels of inhibition were comparable to those obtained with the low molecular weight form isolated and purified directly from the starting materials. This finding seemed to indicate that the conversion involved no major change in the primary pocket specificity.

Dansyl fluoride-urokinase, convertedform The high molecular weight form of urokinase can be transformed into another active species either autocatalytically or by incubation with reducing agents [4,16], findings confirmed recently by Ascenzi et al. [7]. Since the 'converted' form appears to be very similar to the low molecular weight 33 kDa urokinase, we were interested to see whether placing a fluorescent reporter group such as dansyl fluoride near the 'primary' binding pocket of these serine proteases could clarify the structural identity of various forms. Such informa-

Fluorescence parameter Fig. 3 depicts corrected fluorescence spectra for D G N L - and dansyl fluoride-urokinase in aqueous buffers and in buffers prepared with deuterium oxide. Some of the fluorescent parameters are summarized in Table I. Emission spectra taken in deuterium oxide show a considerable increase in fluorescence intensity and quantum yield. This is particularly noticeable for the dansyl fluoride derivatives of urokinase. This deuterium oxide effect is still unexplained theoretically [30-32] but clearly

i

i

i

1 A

B

1

J

J 5OO Wavelength, N M

Wavelength. N M

Fig. 3. Corrected fluorescenceemission spectra in water and deuterium oxide of the dansyl group in derivatives of urokinase. Excitation was at the long wavelengthmaximum of the dye. Excitationbandwidth was 7.5 nm, emission bandwidth was 2.5 nm. (A) Low molecular weight form urokinase, 33 kDa urokinase; (!) dansyl fluoride-33 kDa urokinase in deuterium oxide; (2), dansyl fluoride-33 kDa urokinase in water; (3), DGNL-33 kDa urokinase in deuterium oxide; (4), DGNL-33 kDa urokinase in water. (B) High molecular weight form urokinase, 52 kDa urokinase; (1), dansyl fluoride-52 kDa urokinase in deuterium oxide; (2), dansyl fluoride-52 kDa urokinase in water; (3), DGNL-52 kDa urokinase in deuterium oxide; (4), DGNL-52 kDa urokinase in water. The samples were dissolved in potassium phosphate buffer, 0.1 M, pH 7.0 (p2H 7.0), prepared either with water or deuterium oxide.

409

gives a measure of the exposure of the chromophore. N o shifts in energy spectrum were noted and shape and position of the spectrum remained unchanged with deuterium oxide as solvent. Table I also contains the measured lifetime for the five fluorescent derivatives which have been prepared in this study. Samples inactivated with dansyl fluoride (the first three in Table I) were fully corrected for the exciting light pulse and deconvoluted using a program developed at the Max-Planck-Institute for Biophysical Chemistry, Gtttingen, F.R.G. As indicated in the legend to Fig. 1, two lifetimes were found for these dansyl derivatives. We have assigned in this communication the longer lifetime to the fluorescent species bound to the protein, an assumption supported by a number of publications in the recent literature. A detailed analysis of our time-resolved nanosecond measurements is in progress and will be presented elsewhere. The remaining two samples were measured with a different experimental procedure and without deconvolution analysis of the decay parameters. Therefore the absolute lifetime values cannot be compared directly. For the purpose of the present study only ratios of lifetimes obtained in H 2 0 and 2 H 2 0 under otherwise identical experimental conditions will be considered. Conclusions drawn from such values should therefore be valid. For example, in Table II comparison of the ratios of three and for some cases four

different fluorescent parameters shows reasonable agreement. It is obvious that the same fluorescent probe reflects two quite different environments in the two sets of derivatives. N o changes in such parameter ratios were observed when comparing direct fluorescence (excitation at 330 nm) with sensitized fluorescence (excitation at 280 nm). The overall tertiary structure of the modified proteins has very likely not been altered greatly by exposure to deuterium oxide, based on the rationale that any non-symmetrical variation in the overall structure would be reflected in a changed dipole-dipole energy transfer pattern (Foerster transfer) from the tryptophan matrices to the single dansyl-chromophore acceptor group.

Chromophore accessibility The observed solvent effect on the fluorescence of urokinase derivatives (Table II) can be correlated with the availability of the bound chromophores to interact with the solvent, giving a measure of accessibility. Such information in turn can provide insight into shape and size of the binding pocket which harbors the molecular probe. If no isotope effect can be demonstrated, the probe is probably shielded fully from access to solvent, suggesting the presence of crevice, penetrating relatively deeply into the protein matrix. On the contrary, an almost 100% isotope effect would

TABLE I SPECTROSCOPY CHARACTERISTICS OF DANSYL DERIVATIVES OF DIFFERENT FORMS OF UROKINASE IN H20 AND ZH20 All spectra were taken on a FICA 55 absolute fluorimeter. The lifetime data for the DGNL-urokinases were obtained by excitation with a nitrogen-laser and are uncorrected for the excitation pulse width (4 ns) and not deconvoluted. The absolute lifetime values should therefore not be compared directly with the other values. However,the 2H20/H20 ratios should be valid. DF-UK, urokinase modified with dansyl fluoride; DGNL-UK, urokinase modified with Dns-Giy-Nle-Lys-CH2Ci. Samples

DF-UK 33 DF-UK 33 C DF-UK 52 DGNL-UK 33 DGNL-UK 52

Max. excitation (h, nm)

Max. emission (h, nm)

Lifetime (ns)

H20

2H20

H20

2H20

H20

2H20

315--3 315---3 315±3 322-----3 322 ± 3

315-----3 315---+3 315±3 322-----3 322---3

515-----2 515-'-2 515±2 570-----2 570---+2

515-----2 515---2 515±2 570-----2 570---2

12.6 13.2 12.9 8.5 7.6

20.2 19.9 20.5 10.5 10.2

410 T A B L E II E F F E C T OF D E U T E R I U M O X I D E ON F L U O R E S C E N T P A R A M E T E R S O F SOME U R O K I N A S E DERIVATIVES Fully corrected spectra were taken on a FICA 55 absolute fluorimeter. Lyophilized samples were taken up in potassium phosphate buffer, 0.1 M, pH 7.0 (p2H 7.0) either prepared with H 2 0 or with 2H20. Excitation of samples at 330 nm, bandwidth 7.5 nm, temperature controlled at 20°C. Special care was taken to record the spectra under identical conditions. The emission maxima and the shape of the spectra were identical for a particular pair of derivatives. Lifetime measurements for DGNL-urokinases were obtained with a nitrogen-laser pulse (pulse width, 4 ns) and were not deconvoluted. For abbreviations see Table I. Sample

D F - U K 33 D F - U K 33C D F - U K 52 D G N L - U K 33 D G N L - U K 52

2H20/H20

ratios of several parameters

Means-+ S.D.

Lifetime

Intensity

Q u a n t u m yield

Excitation

1.60 1.51 1.59 1.24 1.34

1.57 1.49 1.63 1.11 1.18

1.60 1.51 1.47 1.04 1.10

1.59 1.50 1.67 N.D. N.D.

imply that the chromophore is freely accessible to solvent and there is no binding pocket. To make such assessments, the enhancement effect of deuterium oxide on model compounds was investigated by measuring relative quantum yields in water/deuterium oxide mixtures. We have previously employed dansyl amide and dansyl ethyl ester as model compounds [33] and have extended these studies to include aqueous mixtures of DnsGly-Nle-Lys-CH2Cl. The water-solubility of the peptide chloroketone allowed us to eliminate the influence of a possible organic solvent effect which might have played a small role in earlier values obtained from model compounds. The results are shown in Fig. 4. The solid line represents an exponential curve fitting of our data points. The ratios which we have obtained from the probemodified urokinase derivatives (Table II) can be converted to percent accessibility. In Table III the results of this conversion have been collected. The two sets of derivatives have clearly different solvent accessibilities. In the dansyl fluoride derivatives the probe seems to be located in a pocket, with one side of the chromophore exposed to the solvent environment, whereas in the peptide chloroketone derivative the chromophore appears to be more deeply embedded in the protein structure. Further support for these proposed differences in structural features comes from evaluation of the apparent polarity of the environment of the probes.

1.59-+0.01 1.50+0.01 1.59 -+ 0.09 1.13-+0.10 1.21 -+0.12

Binding-site polarity Table I and Fig. 3 show that the energy spectra of the two sets of derivatives are quite different. The spectra of dansyl fluoride-urokinase samples are shifted strongly to the blue. We have correlated the emission maxima with the empirical solvent parameter of Kosower [34] and summarized the results in Table IV, which includes newer values obtained with Dns-Gly-Nle-LysCH2C1 in four solvents. The Z value for dioxane was calculated from a reported Et(30 ) value of

o o

1.6

Z 1.2 20

40 6'0 e/o 2H20 , v/v

80

Fig. 4. Effect of deuterium oxide upon fluorescence q u a n t u m yield of D G N L - C M in solutions made up of mixtures of potassium phosphate buffer, 0.1 M, pH 7.0, and identical buffer prepared with deuterium oxide. Excitation of chromophore at 330 nm; excitation and emission bandwidths were 7.5 nm. Temperature controlled at 20°C. The line represents an exponential fit through the experimental points (only a few are shown) with a coefficient of determination of 0.98.

411

chloroketone-modified species experiences a relatively polar milieu.

T A B L E III S O L V E N T ACCESSIBILITY O F T H E D A N S Y L - C H R O M O P H O R E IN SEVERAL F L U O R E S C E N T U R O K I N A S E DERIVATIVES

Discussion

The values are derived from Fig. 4 and Table II. Several model calibration curves obtained at different times and with different solutions were utilized for the reported average values. Previously determined 2 H 2 0 / H 2 0 ratios for dansyl ethyl ester and dansyl amide (Vaz and Schoellmann, Biochim. Biophys. Acta (1976) 439, 206-218) were also considered. Abbreviations, see Table I. Samples

Our results with the dansyl probe, placed inside or close to the binding pocket of three forms of urokinase, indicate a common structural feature in the active sites of all three forms. From the considerable exposure of the chromophore to solvent with an accessibility of almost 70%, the binding pocket appears to be either quite shallow or, alternatively, to be endowed with substantial flexibility, thus facilitating solvent exchange, which could account for the observed effect. The very strong apolar nature of the site with an extrapolated Z value of 57 kcal/mol, which we found identical in all three samples, is intriguing. For dansyl trypsin we have reported a Z value of less than 76.7 [33], but based on the measured emission maxima of 537 nm for trypsin vs. 515 nm for all three forms of urokinase the emission maxima in urokinase are blue-shifted (lower wavelength), corresponding to a shift to more apolar environments. Identical low emission maxima of 510 nm have been found for human a- and 3,-thrombins covalently labeled with dansyl fluoride [13]. Rodier [14] observed a similar blue shift for the anthraniloyl group inserted into the active site of plasmin as

Est. solvent accessibility

(%±S.D.) D F - U K 33 D F - U K 33 C D F - U K 52 D G N L - U K 33 D G N L - U K 52

67.1 ± 6 . 0 60.5+-5.6 69.3--6.5 18.4± 1.8 25.9±3.1

dioxane [35] and from a Z-Et(30 correlation diagram published by Kosower [36]. As in the accessibility study, the probe senses two totally different environments. The very low Z value of the dansyl fluoride-derivatives implies a highly apolar environment for the chromophore close to the primary binding site of urokinase, whereas the more distant dansyl group in the

T A B L E IV E S T I M A T I O N OF P O L A R I T Y OF B I N D I N G SITES IN SOME U R O K I N A S E DERIVATIVES. M A X I M U M OF EMISSION IN W A V E N U M B E R S PF (/~m-~) A N D K O S O W E R ' S Z-VALUES ( K C A L / M O L ) The wavelength maxima of emission are expressed in wavenumbers (/~m - t ) for direct comparison with a previously published, more extensive table of correlation of model compounds with some empirical solvent parameters (Vaz and Schoellmann (1976) Biochim. Biophys. Acta 439, p. 210, Table I). The Z values for the urokinase derivatives were obtained by calculation from the model c o m p o u n d s assuming an exponential fit of the data points (correlation factor, 0.96). The Z value of dioxane was estimated from an Et(30)-Z correlation diagram [36]. Rodier [14] reports a Z value of 74 for dioxane but, using his polynomial fitting function and his own data, we have calculated a Z value of 50. Solvent

DGNL-CM PF

H 2° 2H 2 0 Ethanol Dioxane

1.695 1.709 1.835 1.942

Z value

94.6 93.0 79.6 58.5

D F - U K 33 D F - U K 33 C D F - U K 52

D G N L - U K 33

UF

Z value

1.942 1.942 . .

57.0 57.0 . .

. .

. .

PF

Z value

1.754 1.754

87.6 87.6

412

compared to trypsin, so that a very strong apolar microenvironment close to the primary binding pocket has been demonstrated for at least three samples of trypsin-like blood proteases. In all cases the apparent hydrophobicity is greater than that of trypsin. It remains to be seen whether this is a general, perhaps unique and functionally required feature of some other trypsin-like plasma proteases as well. Conversely, the binding pocket assessed by the chromophore seems to be somewhat larger or more flexible than in trypsin. A recent report on the interaction of aliphatic ammonium and amidinium ions with the two enzymes suggested that both enzymes have a hydrophobic pocket of about equal length and that the primary binding site of urokinase seems less rigid that that of trypsin [8]. A similar conclusion was drawn from a comparative study of sulfonylation with several serine proteases [37]. We have found no significant differences in accessibility and polarity of the primary binding site of the high and low molecular weight forms of urokinase, including the converted form. Thus, any possible differences must be due to dissimilarities in the extended binding regions. The same conclusion was reached by Ascenzi et al. [7], who compared rates of hydrolysis of Z-Lys-pnitrophenyl ester by the two forms of urokinase. We also found that the primary binding pocket S t of the converted form of urokinase, prepared from the high molecular weight form, is identical with that of the directly isolated low molecular weight form. These forms thus appear to be structurally identical, as judged from their catalytic site, suggesting that the high molecular weight form is a direct precursor of the low molecular weight form and probably resulting in general from proteolytic conversion. We detected a remarkably low solvent accessibility of the dansyl-chromophore of about 18% and 26% for the low and high molecular weight forms of DGNL-urokinase combined with relatively polar surrounding of the probes. This finding is somewhat difficult to reconcile because one tends to associate a lack of surface-related solvent exchange with protection in a relatively apolar interior protein matriX. The problem has been discussed briefly elsewhere [33]. Moreover, ideas expressed in the recent literature [38,39] seem to

indicate some inherent difficulties in the interpretation of polarity versus accessibility measurements. As is well known, the action of urokinase and other plasminogen activators is highly specific. A fragment of plasminogen, 38 residues long and encompassing the region normally cleaved by urokinase during activation of the zymogen, is not hydrolyzed at all by urokinase [40]. In a study of susceptibility of urokinase to affinity labeling by peptides of arginine chloromethyl ketones, tripeptide analogues proved more effective than the corresponding tetrapeptide analogues [6]. The critical residue in both cases appeared to correspond to the P4 position, which in the case of the physiological substrate is a cystine residue. A wide variety of residues is acceptable in positions P2 and P3 with a preference for a glycine residue in the P2 position. On the other hand, in position P4 a recognition pattern seems to emerge which is no longer congruent with the linear sequence of the peptide chain (position P4 and higher). Rather, it is likely that additional residues or other structural features situated in the immediate vicinity in the native secondary and/or tertiary structure are being recognized by the enzyme. This extended binding region might well be an essential feature contributing to the high degree of specificity and selectivity of plasminogen activators. Although it is impossible to describe any detailed structural feature of the S4 subsite of urokinase, the site with which the dansyl chromophore is most probably interacting, we propose the existence of a specific binding pocket, penetrating rather deeply into the protein matrix, either preexisting or formed by 'closing-in' on the ligand at the time of interaction. A water molecule or some other polar residue(s) might be part of the feature of this subsite. Various reports in the literature [27,41,42] support the existence of such a site by observing restrictions induced by a group at position P4 implicating structural configurations reciprocating the enzymic subsite S4, as yet unrecognized but in our opinion assessed by the fluorescent probe. A slight difference found in solvent accessibility of the two forms of urokinase might indicate a structural difference in the extended binding region of the two forms. The polarity of the crevice

413

remain unchanged, but the binding pocket was somewhat larger in the high molecular weight form. Our findings corroborate reports of differences in relative enzymic activities of high and low molecular weight forms of urokinase. Such conclusions were reached from a detailed kinetic analysis using various structural forms of plasminogens [43,44]. Differences in the rate of inhibition of high and low molecular weight forms of urokinase by plasma inhibitors have also been reported [45].

Acknowledgements This research was supported in part by U.S. Public Health Service Research Grants HL 23530 and RR05377 from the National Heart, Lung and Blood Institute. We thank Dr. Thomas Jovin, Max-Planck-Institute of Biophysical Chemistry for the kind hospitality during a stay of one of us (G.Sch.) in Goettingen, Germany.

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