Lövgren J, Airas K, Lilja H
Department of Laboratory Medicine, Lund University, Malmö, Sweden.
Eur J Biochem. 1999 Jun;262(3):781-9. doi: 10.1046/j.1432-1327.1999.00433.x.
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 Zn2+ and different protease inhibitors to inhibit hK2 by kinetic measurements. Inhibitory constants were determined for the most effective inhibitors PCI and Zn2+. The high affinity of PCI for hK2 (kass = 2.0 x 10(5) M-1 x s-1) and the high concentrations of PCI (4 microM) and hK2 (0.2 microM) in seminal plasma make hK2 a very likely physiological target protease for PCI. hK2 is inhibited by Zn2+ 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 Zn2+ 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.
人腺激肽释放酶2（hK2）是一种由前列腺表达的丝氨酸蛋白酶，其一级结构与前列腺特异性抗原（PSA）有80%的同源性。最近研究表明，hK2在体外可激活PSA的酶原形式（proPSA），可能是前列腺中PSA的生理激活剂。hK2还能够激活尿激酶并有效裂解纤连蛋白。我们研究了hK2的底物特异性以及前列腺和精浆中存在的锌和细胞外蛋白酶抑制剂对其活性的调节作用。通过确定hK2在精液中的主要凝胶蛋白、精液凝胶蛋白I和II中的切割位点，以及测量各种三肽氨基甲基香豆素底物的水解情况，研究了其酶活性和底物特异性。HK2在单精氨酸或双精氨酸的C末端切割底物。在切割位点的其他几个N末端位置偶尔也会发现碱性氨基酸。因此，hK2的底物特异性与蛋白质前体加工酶的底物特异性非常吻合。通过动力学测量测试Zn2+和不同蛋白酶抑制剂抑制hK2的能力，研究了可能的调节机制。确定了最有效的抑制剂PCI和Zn2+的抑制常数。PCI对hK2的高亲和力（kass = 2.0 x 10(5) M-1 x s-1）以及精浆中高浓度的PCI（4 microM）和hK2（0.2 microM）使得hK2很可能是PCI的生理靶标蛋白酶。hK2在微摩尔浓度下就被Zn2+抑制，该浓度远低于前列腺中发现的9 mM锌浓度。hK2的酶活性可能在前列腺液中受到Zn2+的可逆调节。在前列腺炎（CAP）和晚期转移性癌症中，这种调节可能会受损，导致对hK2活性缺乏控制，从而需要其他控制手段。
