Human glandular kallikrein 2 (hK2) is a serine protease expressed by the prostate gland with 80% identity in primary structure to prostate-specific antigen (PSA). Recently, hK2 was shown to activate the zymogen form of PSA (proPSA) in vitro and is likely to be the physiological activator of PSA in the prostate. hK2 is also able to activate urokinase and effectively cleave fibronectin. We studied the substrate specificity of hK2 and regulation of its activity by zinc and extracellular protease inhibitors present in the prostate and seminal plasma. The enzymatic activity and substrate specificity was studied by determining hK2 cleavage sites in the major gel proteins in semen, semenogelin I and II, and by measuring hydrolysis of various tripeptide aminomethylcoumarin substrates. HK2 cleaves substrates C-terminal of single or double arginines. Basic amino acids were also occasionally found at several other positions N-terminal of the cleavage site. Therefore, the substrate specificity of hK2 fits in well with that of a processor of protein precursors. Possible regulation mechanisms were studied by testing the ability of Zn 2+ and different protease inhibitors to inhibit hK2 by kinetic measurements. Inhibitory constants were determined for the most effective inhibitors PCI and Zn 2+ . The high affinity of PCI for hK2 (k ass = 2.0 Â 10 5 m 21´s21 ) and the high concentrations of PCI (4 mm) and hK2 (0.2 mm) in seminal plasma make hK2 a very likely physiological target protease for PCI. hK2 is inhibited by Zn 2+ at micromolar concentrations well below the 9 mm zinc concentration found in the prostate. The enzymatic activity of hK2 is likely to be reversibly regulated by Zn 2+ in prostatic fluid. This regulation may be impaired in CAP and advanced metastatic cancer resulting in lack of control of the hK2 activity and a need for other means of control.

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The velocity of the pantothenase-catalysed hydrolysis of pantothenate was studied over pH5.5-9, and in the presence of oxalate or oxaloacetate as an inhibitor. The pH-dependence of the reaction can be described by a kinetic equation containing two ionizations of the enzyme, with one ionizable group located at the substrate-binding site, and the other at the inhibitor-binding site. The K,, value of pantothenase to pantothenate depends on the buffer used, and phosphate tends to give somewhat lower values than other buffers. Km also depends on pH, the best activities being observed at basic pH values. The pH-independent Km is 7.6mM in phosphate buffer at 20°C; the corresponding KR " value at pH7 is 15mM. The pK value of the ionizable group at the substrate-binding site was measured by two methods: from the pH-rate profile and from the pH-Km profile. pK is 7.0 in phosphate buffer at 20°C, ranging in various buffers between 6.9 and 7.3. The van't Hoff enthalpies of substrate binding and H+ ion binding were -14kJ/mol and -50kJ/mol respectively. The inhibition by oxalate or oxaloacetate is of non-competitive type and depends on pH, the inhibitors being effective at acidic pH values. The pK value of the ionizable group at the inhibitor-binding site was derived from the measurements of the K, values over the pH range 6-7.5. The pK value was 6.4 in oxaloacetate inhibition, the pH-independent K, being 0.36mM, and the corresponding Ka,' about 1.8mM at pH7. Phenylmethanesulphonyl fluoride was capable of inactivating pantothenase.PantothenasefromPseudomonasfluorescens(pantothenate amidohydrolase, EC 3.5