A fluorescent study of urokinase using active-site directed probes

Abstract

Human high and low molecular weight forms of urokinase (EC 3.4.21.31) were covalently labeled with two active-site directed fluorescent probes, dansyl fluoride and Dns-Gly-Nle-Lys-CH 2CL. Highly purified samples of both derivatives were obtained using affinity chromatography with Sepharose 4 B- ϵ-aminocaproyl- p-aminobenzamidine and Sepharose 4B-ϵ-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. Fiuorescence 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and deuterium oxide with a photo-counting technique and the decay data were analyzed by a method developed by Striker (unpublished data).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 Schoellmann, G. (1976) Biochim. Biophys. Acta 439, 206–218) and with the new model compound Dns-Gly-Nle-Lys-CH 2CI 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 S 4, 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 S 4 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 (Wohl, R.C., Summaria, L. and Robbins, K.L. (1980) J. Biol. Chem. 255, 2005).