The kinetics of a series of Glu-plasminogen ligand-binding processes were investigated at pH 7.8 and 25 degrees C (in 0.1 M-NaCl). The ligands include compounds analogous to C-terminal lysine residues and to normal lysine residues. Changes of the Glu-plasminogen protein fluorescence were measured in a stopped-flow instrument as a function of time after rapid mixing of Glu-plasminogen and ligand at various concentrations. Large positive fluorescence changes (approximately 10%) accompany the ligand-induced conformational changes of Glu-plasminogen resulting from binding at weak lysine-binding sites. Detailed studies of the concentration-dependencies of the equilibrium signals and the rate constants of the process induced by various ligands showed the conformational change to involve two sites in a concerted positive co-operative process with three steps: (i) binding of a ligand at a very weak lysine-binding site that preferentially, but not exclusively, binds C-terminal-type lysine ligands, (ii) the rate-determining actual-conformational-change step and (iii) binding of one more lysine ligand at a second weak lysine-binding site that then binds the ligand more tightly. Further, totally independent initial small negative fluorescence changes (approximately 2-4%) corresponding to binding at the strong lysine-binding site of kringle 1 [Sottrup-Jensen, Claeys, Zajdel, Petersen & Magnusson (1978) Prog. Chem. Fibrinolysis Thrombolysis 3, 191-209] were observed for the C-terminal-type ligands. The finding that the conformational change in Glu-plasminogen involves two weak lysine-binding sites indicates that the effect cannot be assigned to any single kringle and that the problem of whether kringle 4 or kringle 5 is responsible for the process resolves itself. Probably kringle 4 and 5 are both participating. The involvement of two lysine binding-sites further makes the high specificity of Glu-plasminogen effectors more conceivable.

Bmdtnil of 6-ammohexaaolu acid tO the AH.I)Ie, a weak lyline bittdln8 lib: tn Glu,plasmlnoilen, =liters the conformation of tile moleculc~ The kmel. ice= of the blndlnii and the ac=:ompanylnil ¢onformational chanlle are investljated at pH 7 I!, 2.~'C Chanlle~ of lama,tie protein iluor~ccnee ~ete measured as a function of time after raped mixing in a stopped.flew app*ratus, The results reflect a two.step ra~¢tto,t meehan,sm. Rapid ass~iation of Glu.plasmtnogen and 6-ammohe~anoi¢ nod (K) =,44 raM) followed by the conform.tional change (k= =69 (-= and k ,~ ,,. 3 s =1 with an overall dissc

No abstract

The initial rate steady-state kinetics of carboxypeptidase-Y-catalyzed hydrolysis and aminolysis reactions with some a-N-benzoyl-L-tyrosinyl compounds has been investigated using L-valinamide as the nucleophile in aminolysis. Hydrolysis of a-N-benzoyl-L-tyrosinyl ethyl ester, 4-nitroanilide, and -amide has been studied in the pH range 4 -9. The results are interpreted in terms of the classical serine proteinase mechanism, which involves enzyme-substrate complex formation, followed by acylation and deacylation of the enzyme. The three reactions share the same deacylation step. It is rate-determining with the ester substrate, but with the 4-nitroaniline acylation is and this is even more pronounced with the amide. From the pH dependencies, no change of rate-determining step is apparent in the range pH 4-9. For the 4-nitroanilide and the amide substrates, the kinetic parameter, k,/K,, is influenced by an ionizing group with a pK value of 6. Probably this is the active-site histidine residue, which thus is active in acylation in its deprotonated form. That group affects the deacylation reaction similarly as seen from the kinetics of the ester substrate.Aminolysis occurs in parallel to hydrolysis in the presence of reactive nucleophiles. Here Lvalinamide was used as model nucleophile. The analysis of the observed kinetic eflects of L-valinamide on the initial rate behaviour of carboxypeptidase-Y-catalyzed hydrolysis reactions suggests a reaction mechanism which involves (a) the binding of the free nucleophile to the free enzyme and (b) reaction of the free nucleophile with the acyl-enzyme complex forming an enzyme-aminolysis product complex, which dissociates into the free enzyme and the aminolysis product. The reactions are characterized by a number of kinetic parameters, the values of which are determined.The results of aminolysis progress reactions indicate that the formation of the product in high yields is strongly dependent on the leaving group of the substrate. The initial production of aminolysis product, however, is the same for the three substrates. But the fact that their kJKm values differ by several orders of magnitude leads to significantly different progresses of the aminolysis. The ester substrate is the only one that efficiently competes with and hinders the hydrolysis of the aminolysis product.Carboxypeptidase Y from baker's yeast belongs to the group of serine carboxypeptidases. These enzymes are functionally similar, but structurally unrelated to the serine endopeptidases (Dreddam, 1986;Liao and Remington, 1990). Carboxypeptidase Y catalyzes the hydrolysis of peptide and ester bonds and exhibits broad substrate specificity (Bai et al., 1975;Hayashi et al., 1975).Recently, the enzyme has been successfully used in enzymic peptide synthesis (Widmer and Johansen, 1979;Hellio et al., 1988). The hydrolysis of substrates apparently proceeds via the classical serine protease mechanism (Quellet and Stewart, 1959) which involves enzyme-substrate complex formation followed by acylation and deacylation of the...