ABSTRACT
Interaction between duodenase (a granase family member) from bovine duodenal mucosa and recombinant antichymotrypsin (rACT) and its P1 variants has been studied. Association rate constants (k(a)) were 11, 6.8, and 17 mM(-1).sec(-1) for rACT, ACT L358M, and ACT L358R, respectively. Natural antitrypsin (AT) compared to ACT was a 20 times more effective duodenase inhibitor (in terms of k(a)). Duodenase interacted with P1 variants of ACT via a suicide mechanism with stoichiometry of the process SI = 1.2. The nature of the P1 residue of the inhibitor did not influence the interaction if other residues did not meet conformational requirements of the duodenase substrate-binding pocket. Also, interaction of duodenase with ACT variants containing residues from AT reaction center loop (rACT P2-P3', rACT P3-P4', rACT P4-P3', and rACT P6-P4') was studied. The inhibition type ([E](0) = 1.10(-7) M, 25 degrees C) was revealed to be reversible-like, and efficacy of inhibition decreased with increase in the substituted part of the reactive center loop. Constants of inhibition (K(i)) were measured. Efficacy of interaction between the enzyme (duodenase) and inhibitor depends on topochemical correspondence between a substrate-binding pocket of the enzyme and substrate structure.
Subject(s)
Serine Endopeptidases/chemistry , Serine Proteinase Inhibitors/chemistry , alpha 1-Antichymotrypsin/chemistry , Animals , Binding Sites , Cattle , Kinetics , Molecular Structure , Protein Binding , Serine Endopeptidases/genetics , Serine Proteinase Inhibitors/genetics , alpha 1-Antichymotrypsin/geneticsABSTRACT
Interaction between a serine proteinase from bovine duodenum and human serum alpha(2)-macroglobulin (alpha(2)-MG) was studied. alpha(2)-MG is established to be one of the most effective duodenase inhibitors. The enzyme is completely inhibited in less than 30 sec at equimolar ratio of the inhibitor and enzyme (concentration 2 x 10(-8) M). Under identical conditions, the rate of duodenase association with alpha(2)-MG is at least 2.5-fold higher than the rate of chymotrypsin association with this inhibitor. The interaction with duodenase results in proteolysis of the inhibitor subunit in the "bait region". Similarly to other proteases, duodenase in the complex with alpha(2)-MG retains the intact catalytic apparatus and ability to hydrolyze some small substrates. But the duodenase-inhibitor complex is fully inactive to proteins (bovine serum albumin). The stoichiometry of the enzyme interaction with the inhibitor is 2 : 1 (mol/mol). Based on the association rate constant and the termination time of the duodenase and alpha(2)-MG in vivo association, alpha(2)-MG is suggested to be a physiological regulator of the enzyme.
Subject(s)
Serine Endopeptidases/metabolism , Substrate Specificity , alpha-Macroglobulins/metabolism , Animals , Catalytic Domain , Cattle , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Humans , Kinetics , Molecular Weight , Multiprotein Complexes/metabolism , Protein Binding , Serine Proteinase Inhibitors/metabolismABSTRACT
The inhibition of exogenous serine proteinases of different origin by cationic protease inhibitors BWI-1c, -2c, -3c, and -4c from buckwheat (Fagopyrum esculentum Moench) seeds has been studied. High efficiency of the inhibitors in binding bovine trypsin and chymotrypsin as well as their broad antiprotease effect, including inhibition of proteinases secreted by fungi and bacteria, has been demonstrated. According to the data obtained, it is proposed that cationic inhibitors from buckwheat seeds may participate in the defense of plants against fungal and bacterial infection.
Subject(s)
Fagopyrum/chemistry , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/isolation & purification , Serine Proteinase Inhibitors/pharmacology , Animals , Bacteria/enzymology , Cattle , Chymotrypsin/antagonists & inhibitors , Chymotrypsin/metabolism , Fungi/enzymology , Seeds/chemistry , Serine Proteinase Inhibitors/chemistry , Subtilisin/antagonists & inhibitors , Subtilisin/metabolism , Trypsin/metabolismABSTRACT
The interaction of duodenase, a new serine protease from a small group of Janus-faced proteases, with serpins, alpha 1-protease inhibitor (alpha 1-PI) and antichymotrypsin (ACT) from human blood serum, was studied. The stoichiometry of the inhibition process was found to be 1.2 and 1.3 mol/mol for alpha 1-PI and ACT, respectively. The presence of a stable enzyme-inhibitory complex duodenase-alpha 1-PI was confirmed by SDS-PAGE. No formation of the duodenase-ACT complex was demonstrated; instead, the band of the cleaved inhibitor was indicated upon the ACT hydrolysis. The suicide mechanism of the duodenase interaction with the human blood serpins was proved. The association rate constants (Ka, M-1 s-1) were 2.4 +/- 0.3 x 10(5) for alpha 1-PI and 3.0 +/- 0.4 x 10(5) for ACT. These results indicate the possibility of the regulation of duodenase activity by endogenous serpins. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.
Subject(s)
Serine Endopeptidases/metabolism , Serpins/blood , Electrophoresis, Polyacrylamide Gel , Humans , KineticsABSTRACT
The interaction between duodenase, a newly recognized serine proteinase belonging to the small group of Janus-faced proteinases, and alpha1-proteinase inhibitor (alpha1-PI) from human serum was investigated. The stoichiometry of the inhibition was 1.2 mol/mol. The presence of a stable enzyme-inhibitor complex was shown by SDS-PAGE. The mechanism of interaction between duodenase and alpha1-PI was shown to be of the suicide type. The equilibrium and inhibition constants are 13 +/- 3 nM and (1.9 +/- 0.3).105 M-1.sec-1, respectively. Based on the association rate constant of the enzyme-inhibitor complex and localization of duodenase and alpha1-PI in identical compartments, alpha1-PI is suggested to be a duodenase inhibitor in vivo.
