Human α-Thrombin Inhibition by the Active Site Titrant Nα-(N,N-dimethylcarbamoyl)-α-azalysine p-nitrophenyl ester: A Comparative Kinetic and X-ray Crystallographic Study

Human α-thrombin inhibition by the highly selective compounds N-ethoxycarbonyl-d-phe-pro-α-azalys p-nitrophenyl ester and N-carbobenzoxy-pro-α-azalys p-nitrophenyl ester: A kinetic, thermodynamic and x-ray crystallographic study

“…P1 in Scheme I), which is not followed by the steady-state reaction (see Figure 1). According to Scheme I, and in agreement with crystallographic observations (see the present study, and Nardini et al, 1996), the serine proteinase deacylation process is rate limiting for the enzymatic hydrolysis of Dmc-azaLys-ONp. On the other hand, the dissociation of the E:P3 reversible complex appears to be rate limiting for the thrombin catalyzed cleavage of Eoc-D-Phe-ProazaLys-ONp and Cbz-Pro-azaLys-ONp.…”

“…Since thrombin contains one active site per molecule (Bode et al, 1989(Bode et al, , 1992, the active enzyme concentration may be directly determined from the value of p, according to equation (2) (see Figure 1). The enzyme concentration obtained by using Eoc-D-Phe-Pro-azaLys-ONp and Cbz-ProazaLys-ONp is in excellent agreement with that determined: (1) by titrating the same enzyme sample with pN-pGB, Cbz-Arg-ONp, Cbz-Lys-ONp and Dmc-azaLys-ONp Nardini et al, 1996); and (2) spectrophotometrically from the optical absorption at 280 nm (Elion et al, 1986).…”

“…In previous crystallographic and functional studies we showed that acylating compounds based on the a-azalysine substructure could be used as effective inhibitors of serine proteinases, as well as active site titrants (Ascenzi et al, 1995b;Sartori et al, 1995;Balliano et al, 1996;Nardini et al, 1996). Here we extend the analysis to new very speci®c azapeptide p-nitrophenyl esters based on a-azaLys in P 1 , targeted to the active site of thrombin.…”

“…is the apparent ®rst order rate constant of the time course of p-nitrophenol release (Ascenzi et al, 1995b;Sartori et al, 1995;Balliano et al, 1996;Nardini et al, 1996). The time course of p-nitrophenol release for the thrombin catalyzed hydrolysis of Eoc-D-Phe-Pro- azaLys-ONp, Cbz-Pro-azaLys-ONp and DmcazaLys-ONp corresponds to a single exponential decay for more than 95% of its course (see Figure 1).…”

From natural to synthetic multisite thrombin inhibitors

“…27 However, larger deviations were observed in some crystal structures more recently reported. [32][33][34] A five residue loop near Trp 148 defines the lower rim of the active site. This region, often referred to as the autolysis loop, is characterized by a high degree of flexibility, since it exhibits a variety of different or disordered conformations in the known thrombin structures.…”