title: Substrate specificity and inhibition of human kallikrein-related peptidase 3 (KLK3 or PSA) activated with sodium citrate and glycosaminoglycans.
abstract: We report the enzymatic properties and substrate specificity of human recombinant KLK3 in the presence of glycosaminoglycans (GAGs) and sodium citrate. This salt is highly concentrated in prostate and in its presence KLK3 had a similar hydrolytic efficiency as chymotrypsin. In contrast to the latter peptidase, KLK3 activated by sodium citrate efficiently hydrolyzed substrates containing R, H and P at the P1 position. Activated KLK3 also cleaved peptides derived from the bradykinin domain of human kininogen at the same sites as human kallikrein KLK1, but presented low kininogenase activity. Angiotensin I has several sites for hydrolysis by KLK3; however, it was cleaved only at the Y-I bond (DRVY downward arrowIHPFHL). Sodium citrate modulated KLK3 conformation as observed by alterations to the intrinsic fluorescence of phenylalanines and tryptophans. Activated KLK3 was reversibly inhibited by Z-Pro-Prolinal and competitively inhibited by ortho-phenantroline. Together, these are noteworthy observations for the future design of specific non-peptide inhibitors of KLK3 and to find natural substrates.
authors.affiliation: Department of Biophysics, Universidade Federal de São Paulo, Brazil.
authors.forename: Douglas
authors.initials: D
authors.lastname: Andrade
authors.forename: Diego M
authors.initials: DM
authors.lastname: Assis
authors.forename: Aurelio Resende
authors.initials: AR
authors.lastname: Lima
authors.forename: Juliana R
authors.initials: JR
authors.lastname: Oliveira
authors.forename: Mariana S
authors.initials: MS
authors.lastname: Araujo
authors.forename: Sachiko I
authors.initials: SI
authors.lastname: Blaber
authors.forename: Michael
authors.initials: M
authors.lastname: Blaber
authors.forename: Maria A
authors.initials: MA
authors.lastname: Juliano
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
mesh_terms: D000595:Amino Acid Sequence; D000809:Angiotensins / Q000378:metabolism; D002712:Chlorides / Q000737:chemistry / Q000494:pharmacology; D002951:Citrates / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D031541:Fluorescence Resonance Energy Transfer; D006025:Glycosaminoglycans / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007610:Kallikreins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D019728:Kininogen, Low-Molecular-Weight / Q000378:metabolism; D008969:Molecular Sequence Data; D017430:Prostate-Specific Antigen; D011480:Protease Inhibitors / Q000494:pharmacology*; D012492:Salts / Q000737:chemistry / Q000494:pharmacology; D000077559:Sodium Citrate; D013379:Substrate Specificity
chemical_list: D000809:Angiotensins; D002712:Chlorides; D002951:Citrates; D006025:Glycosaminoglycans; D019728:Kininogen, Low-Molecular-Weight; D011480:Protease Inhibitors; D012492:Salts; D000077559:Sodium Citrate; C549870:KLK3 protein, human; D007610:Kallikreins; D017430:Prostate-Specific Antigen