Kinetic Study of the Effect of Heparin on the Amidase Activity of Trypsin, Plasmin and Urokinase

Abstract

SummaryIt was found that the effect of heparin on the amidase activity of urokinase (E C 3.4.21.31), plasmin (E C 3.4.21.7) and trypsin (E C 3.4.21.4) depended on the substrate used. No effect of heparin on the amidase activity of urokinase and trypsin was observed when Pyro Glu-Gly-Arg-p-nitroanilide (S-2444) and α-N-acetyl-L-lysine-p-nitroanilide (ALNA) were used as substrates. Heparin acted as a uncompetitive inhibitor of trypsin (Ki = 1.2×10-6 M), plasmin (Ki = 4.9×10-6 M) and urokinase (Ki = l.0×10-7 M) when Bz-Phe-Val-Arg-p-nitroanilide (S-2160), H-D-Val-Leu-Lys-p-nitroanilide (S-2251) and plasminogen, respectively, were used as substrates. These results, as well as the data obtained by studying the effect of the simultaneous presence of heparin and competitive inhibitors suggest that although heparin is not bound at the active center of these enzymes, it may influence the effectivity of catalysis.

Recent Advances on Plasmin Inhibitors for the Treatment of Fibrinolysis‐Related Disorders

“…These results suggest that heparin induced microscopic environmental changes in the active site of plasmin by binding to an allosteric site . In fact, kinetic studies using chromogenic substrate S‐2251 demonstrated that heparin is a noncompetitive inhibitor of plasmin with a K i of 4.9 μM …”

“…However, increasing the plasmin concentration relative to heparin reduced the rate. 237 Heparin bound directly to plasmin with a K D of 10 nM [238][239][240] and induced a conformational change in its active site. 241 Interestingly, heparin enhanced hydrolysis of several chromogenic substrates through an increase in k CAT (K M unchanged).…”

“…241 In fact, kinetic studies using chromogenic r AL-HORANI AND DESAI substrate S-2251 demonstrated that heparin is a noncompetitive inhibitor of plasmin with a K i of 4.9 μM. 240 Recently, Chander et al reported plasmin inhibition under physiological conditions by a covalent complex of AT and heparin. 235 Results revealed that the increase in plasmin inactivation is brought about by inducing a conformational change in the enzyme.…”

Plasmin Regulation through Allosteric, Sulfated, Small Molecules

“…Yet, the literature supports the idea of allosteric modulation of plasmin's catalytic activity. For example, heparin is known to bind directly to plasmin with a KD of 10 nM and induce a conformational change in its active site by interacting with an allosteric site [22][23][24][25]. Likewise, N-oleoyl heparin was also found to inhibit plasmin (IC50 = 16 nM) and Lineweaver-Burk analysis indicated noncompetitive inhibition mechanism [26].…”

“…The monomeric scaffolds included chalcones (compounds 1-10), flavonoids (11)(12)(13)(14)(15)(16) [30][31][32], sucrose octasulfate (17) [40], quinazolinones (18 and 19) [37], and tetrahydro-isoquinolines (20)(21)(22)(23)(24)(25)(26)(27) [36], whereas the dimeric scaffolds comprised flavonoid-quinazolinone heterodimers (28)(29)(30)(31)(32)(33)(34) [37], bis-quinazolinones homodimers (35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47) [37], and bis-flavonoid homodimers (48-55). In addition to the inherent diversity of the scaffolds in this library, NSGMs also differed in the number (1 to 8) and orientation of the sulfate groups.…”