title: Characterization of the prostate-specific antigen (PSA) catalytic mechanism: a pre-steady-state and steady-state study.
abstract: Prostate-specific antigen (PSA), an enzyme of 30 kDa grouped in the kallikrein family is synthesized to high levels by normal and malignant prostate epithelial cells. Therefore, it is the main biomarker currently used for early diagnosis of prostate cancer. Here, presteady-state and steady-state kinetics of the PSA-catalyzed hydrolysis of the fluorogenic substrate Mu-His-Ser-Ser-Lys-Leu-Gln-AMC (spanning from pH 6.5 to pH 9.0, at 37.0°C) are reported. Steady-state kinetics display at every pH value a peculiar feature, represented by an initial "burst" phase of the fluorescence signal before steady-state conditions are taking place. This behavior, which has been already observed in other members of the kallikrein family, suggests the occurrence of a proteolytic mechanism wherefore the acylation step is faster than the deacylation process. This feature allows to detect the acyl intermediate, where the newly formed C-terminal carboxylic acid of the cleaved substrate forms an ester bond with the -OH group of the Ser195 catalytic residue, whereas the AMC product has been already released. Therefore, the pH-dependence of the two enzymatic steps (i.e., acylation and deacylation) has been separately characterized, allowing the determination of pKa values. On this basis, possible residues are tentatively identified in PSA, which might regulate these two steps by interacting with the two portions of the substrate.
authors: Department of Sciences, University of Roma Tre, Roma, Italy.
authors: Luigi
authors: L
authors: Tomao
authors: Department of Clinical Sciences and Translational Medicine, University of Roma "Tor Vergata", Roma, Italy; Interuniversity Consortium for the Research on Chemistry of Metals in Biological Systems, Bari, Italy.
authors: Diego
authors: D
authors: Sbardella
authors: Department of Clinical Sciences and Translational Medicine, University of Roma "Tor Vergata", Roma, Italy; Interuniversity Consortium for the Research on Chemistry of Metals in Biological Systems, Bari, Italy.
authors: Magda
authors: M
authors: Gioia
authors: Department of Sciences, University of Roma Tre, Roma, Italy; Interdepartmental Laboratory of Electron Microscopy, University of Roma Tre, Roma, Italy.
authors: Alessandra
authors: A
authors: Di Masi
authors: Department of Clinical Sciences and Translational Medicine, University of Roma "Tor Vergata", Roma, Italy; Interuniversity Consortium for the Research on Chemistry of Metals in Biological Systems, Bari, Italy.
authors: Stefano
authors: S
authors: Marini
authors: Department of Sciences, University of Roma Tre, Roma, Italy; Interdepartmental Laboratory of Electron Microscopy, University of Roma Tre, Roma, Italy.
authors: Paolo
authors: P
authors: Ascenzi
authors: Department of Clinical Sciences and Translational Medicine, University of Roma "Tor Vergata", Roma, Italy; Interuniversity Consortium for the Research on Chemistry of Metals in Biological Systems, Bari, Italy.
authors: Massimo
authors: M
authors: Coletta
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D017430:Prostate-Specific Antigen / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D017430:Prostate-Specific Antigen