Enzymatic action of human glandular kallikrein 2 (hK2). Substrate specificity and regulation by Zn2+ and extracellular protease inhibitors.

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.     

Amidolytic activity of prostatic acid phosphatase on human semenogelins and semenogelin-derived synthetic substrates.

In addition to kallikrein hK3, a serine protease generally reported as PSA (prostate-specific antigen), at least two other enzymes in human seminal plasma also cleave synthetic peptidyl substrates derived from the sequence of human semenogelins. We have identified one of these as prostatic acid phosphatase (PAP), a major component of prostatic fluid whose physiological function is unclear. The other is a high Mr basic protein present at low concentrations in seminal plasma and that remains to be characterized. PAP was purified to homogeneity from freshly ejaculated seminal plasma. Its N-terminal sequence and its phosphatase properties (hydrolysis of para-nitrophenylphosphate at low pH) were determined, and its inhibition by sodium fluoride measured. Both purified and commercial PAP also had amidolytic activity on peptide substrates derived from the semenogelin sequence at neutral and slightly basic pH. The k(cat)/K(m) values were in the 10(2)-10(3) m(-1) x s(-1) range using fluorogenic semenogelin-derived substrates whose peptidyl moiety included cleavage sites that had been identified ex vivo. PAP cleavage sites differed from those of hK3 and were mainly at P1 = Gln residues or between residues bearing hydroxyl groups. PAP amidolytic activity was poorly inhibited by all currently used wide spectrum proteinase inhibitors. Only 3-4 dichloroisocoumarin and benzamidine inhibited purified PAP. Purified human semenogelin was cleaved by purified and commercial PAP at neutral pH; the two main cleavage sites were at Tyr292 and Ser170 (semenogelin I sequence), only the former has been identified ex vivo by analysis of seminal plasma.     

Interplay of human tissue kallikrein 4 (hK4) with the plasminogen activation system: hK4 regulates the structure and functions of the urokinase-type plasminogen activator receptor (uPAR)

The plasminogen activation system is involved in cancer progression and metastasis. Among other proteolytic factors, it includes the serine protease urokinase-type plasminogen activator (uPA) and its three-domain (D1D2D3) receptor uPAR (CD87), which focuses plasminogen activation to the cell surface. The function of uPAR is regulated in part through shedding of domain D1 by proteases, e.g., uPA itself or plasmin. Human tissue kallikrein 4 (hK4), which is highly expressed in prostate and ovarian tumor tissue, was previously shown to cleave and activate the pro-enzyme forms of prostate-specific antigen (PSA, tissue kallikrein hK3) and uPA. Here we demonstrate that uPAR is also a target for hK4, being cleaved in the D1-D2 linker sequence and, to a lesser extent, in its D3 juxtamembrane domain. hK4 may thus modulate the tumor-associated uPA/uPAR-system activity by either activating the pro-enzyme form of uPA or cleaving the cell surface-associated uPA receptor.     

Human tissue kallikrein 9: production of recombinant proteins and specific antibodies

Human tissue kallikreins (genes, KLKs; proteins, hKs) are a subgroup of hormonally regulated serine proteases. Two tissue kallikreins, namely hK2 and hK3 (prostate-specific antigen, PSA), are currently used as serological biomarkers of prostate cancer. Human tissue kallikrein 9 (KLK9) is a newly identified member of the tissue kallikrein gene family. Recent reports have indicated that KLK9 mRNA is differentially expressed in ovarian and breast cancer and has prognostic value. Here, we report the production of recombinant hK9 (classic form) using prokaryotic and mammalian cells and the generation of polyclonal antibodies. Total testis tissue mRNA was reverse-transcribed to cDNA, amplified, cloned into a pET/200 TOPO plasmid vector, and transformed into E. coli cells. hK9 was purified and used as an immunogen to generate polyclonal antibodies. Full-length KLK9 cDNA was also cloned in the vector pcDNA3.1 and was expressed in CHO cells. The identity of hK9 was confirmed by mass spectrometry. hK9 rabbit antiserum displayed no cross-reactivity with other tissue kallikreins and could specifically recognize E. coli- and CHO-derived hK9 on Western blots. hK9 was mainly detected in testis and seminal vesicles by Western blotting. The reagents generated here will help to define the physiological role of this tissue kallikrein and its involvement in human disease.     

Inhibition of human kallikreins 5 and 7 by the serine protease inhibitor lympho-epithelial Kazal-type inhibitor (LEKTI)

LEKTI is a 120-kDa protein that plays an important role in skin development, as mutations affecting LEKTI synthesis underlie Netherton syndrome, an inherited skin disorder producing severe scaling. Its primary sequence indicates that the protein consists of 15 domains, all resembling a Kazal-type serine protease inhibitor. LEKTI and two serine proteases belonging to the human tissue kallikrein (hK) family (hK5 and hK7) are expressed in the granular layer of skin. In this study, we characterize the interaction of two recombinant LEKTI fragments containing three or four intact Kazal domains (domains 6-8 and 9-12) with recombinant rhK5, a trypsin-like protease, and recombinant rhK7, a chymotrypsin-like protease. Both fragments inhibited rhK5 similarly in binding and kinetic studies performed at pH 8.0, as well as pH 5.0, the pH of the stratum corneum where both LEKTI and proteases may function. Inhibition equilibrium constants (Ki) measured either directly in concentration-dependent studies or calculated from measured association (kass) and dissociation (kdis) rate constants were 1.2-5.5 nM at pH 8.0 and 10-20 nM at pH 5.0. At pH 8.0, kass and kdis values were 4.7 x 10(5) M(-1) s(-1) and 5.5 x 10(-4) s(-1), and at pH 5.0 they were 4.0 x 10(4) M(-1) s(-1) and 4.3 x 10(-4) s(-1), respectively. The low Ki and kdis values (t1/2 of 20-25 min) indicate tight and specific association. Only fragment 6-9' was a good inhibitor of rhK7, demonstrating a Ki of 11 nM at pH 8.0 in a reaction that was rapidly reversible. These results show that LEKTI, at least in fragment form, is a potent inhibitor of rhK5 and that this protease may be a target of LEKTI in human skin.     

Ejaculates from the common marmoset (Callithrix jacchus) contain semenogelin and beta-microseminoprotein but not prostate-specific antigen

Human seminal plasma contains high concentrations of prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), beta-microseminoprotein (MSP), semenogelin I (SgI), and semenogelin II (SgII), whereas only PAP and MSP are present in rodents. In order to gain a better understanding of the evolution and function of semen proteins, we have studied ejaculates from the common marmoset (Callithrix jacchus)-a New World monkey. Semen samples were analyzed with SDS-PAGE, Western blotting, and isoelectric focusing. Under reducing conditions the dominating protein components appear as heterogeneous material of 55-70 kDa and distinct protein bands of 85, 17, 16, and 15 kDa. The heterogeneous material contains glycosylated material detected by an antiserum recognizing both human SgI and SgII. Southern blotting indicates that the common marmoset has genes for both SgI and SgII. There are several marmoset MSP genes, but the strong immunoreactivity against one 15 kDa semen component with pI 7.3 suggests preferential expression of one gene in the prostate. Expression of two other genes cannot be excluded as indicated by weak reaction to isoforms with pI 6.6 and 4.9. Unexpectedly, PSA was not detected by either immunological methods or activity measurements. This is in agreement with results from Southern blotting suggesting that the common marmoset might not have a PSA gene. Thus, in this study we have shown that semen coagulum proteins are present in marmoset seminal plasma, but the lack of PSA precludes a similar liquefaction as of human semen.     

Expression, purification, and characterization of deglycosylated human pro-prostate-specific antigen

Wild-type and deglycosylated forms of human prostate-specific antigen were expressed in Chinese hamster ovary (CHO) cells as zymogens. ProPSA was collected from conditioned medium and purified using a single cation-exchange chromatographic step for the deglycosylated form and cation-exchange followed by gel filtration chromatography for the wild-type form. Recombinant wild-type proPSA produced in CHO cells has an average MW of 34.5 kDa, whereas the deglycosylated proPSA has a MW of 32.4 kDa. Both forms of proPSA were activated in vitro and the kinetic properties measured for the deglycosylated PSA are very similar to those of the wild-type recombinant PSA and the native PSA isolated from seminal fluid. These results suggest that deglycosylated PSA is likely to be very similar to native PSA with respect to its three-dimensional structure and will provide a homogeneous protein preparation necessary for X-ray crystallographic analysis.     

Specificity profiling of seven human tissue kallikreins reveals individual subsite preferences

Human tissue kallikreins (hKs) form a family of 15 closely related (chymo)trypsin-like serine proteinases. These tissue kallikreins are expressed in a wide range of tissues including the central nervous system, the salivary gland, and endocrine-regulated tissues, such as prostate, breast, or testis, and may have diverse physiological functions. For several tissue kallikreins, a clear correlation has been established between expression and different types of cancer. For example, the prostate-specific antigen (PSA or hK3) serves as tumor marker and is used to monitor therapy response. Using a novel strategy, we have cloned, expressed in Escherichia coli or in insect cells, refolded, activated, and purified the seven human tissue kallikreins hK3/PSA, hK4, hK5, hK6, hK7, hK10, and hK11. Moreover, we have determined their extended substrate specificity for the nonprime side using a positional scanning combinatorial library of tetrapeptide substrates. hK3/PSA and hK7 exhibited a chymotrypsin-like specificity preferring large hydrophobic or polar residues at the P1 position. In contrast, hK4, hK5, and less stringent hK6 displayed a trypsin-like specificity with strong preference for P1-Arg, whereas hK10 and hK11 showed an ambivalent specificity, accepting both basic and large aliphatic P1 residues. The extended substrate specificity profiles are in good agreement with known substrate cleavage sites but also in accord with experimentally solved (hK4, hK6, and hK7) or modeled structures. The specificity profiles may lead to a better understanding of human tissue kallikrein functions and assist in identifying their physiological protein substrates as well as in designing more selective inhibitors.     

Expression and purification of recombinant active prostate-specific antigen from Escherichia coli

Human prostate-specific antigen (PSA), a 33 kDa serine protease with comprehensive homology to glandular kallikrein, is secreted from prostatic tissue into the seminal fluid and enters into the circulation. The level of PSA increases in the serum of patients with prostatic cancer and hence is widely employed as a marker of the disease status. In particular, an enzymatically active PSA that is a form cleaved at the N-terminal seven-amino-acids prosequence, APLILSR, of proPSA may play an important roll in the progression of prostate cancer. Thus, the presence of the active form would selectively discriminate the cancer from benign prostatic hyperplasia. In this study, we developed a convenient purification method for the acquisition of active PSA and proPSA. Recombinant proPSA and active PSA were expressed directly in Escherichia coli, easily and efficiently isolated from inclusion bodies, refolded, and purified. Moreover, the enzymatic activity of the recombinant active PSA was confirmed as serine protease using chromogenic chymotrypsin substrate. This purified active PSA could be further applied to scrutinize the biological or conformational characteristics of the protein and to develop specific diagnostic and/or therapeutic agents against prostate cancer.     

Secretion of endogenous kallikreins 2 and 3 by androgen receptor-transfected PC-3 prostate cancer cells

Androgen independent PC-3 cells lack androgen receptor (AR) expression and do not produce kallikrein 2 (hK2) or 3 (prostate-specific antigen, PSA). In this paper, we examined the ability of androgens to stimulate PSA and hK2 production in AR transfected PC-3 cells (PC-3(AR)) and compared this to LNCaP cells. PSA and hK2 were measured in the culture medium and cell lysates using an ELISA-based immunofluorometric assay. Only androgens were able to induce PSA and hK2 secretion in PC-3(AR) cells in a dose- and time-dependent manner depending on the level of AR present. The level of androgen-induced PSA and hK2 secretion in PC-3(AR) cells was approximately 1.5 and 0.9% that induced in LNCaP cells, respectively. Insulin-like growth factor-I (IGF-I), which has been shown to activate AR in the absence of ligand, did not activate PSA secretion in the absence of androgen, but further increased the dihydrotestosterone-induced PSA secretion in PC-3(AR) cells. The lack of PSA and hK2 production in parental PC-3 cells is thus a result of their lack of AR expression. PSA and/or hK2 production in PC-3(AR) cells can thus serve as an endogenous reporter system to investigate AR action or to screen putative endocrine disrupters.     

人激肽释放酶-1在甲醇酵母中高水平表达、纯化与鉴定

将自肾脏cDNA中获得的人激肽释放酶-1(Human kallikrein 1, hK1)基因插入到pPICZαA载体中, 构建甲醇酵母分泌型表达载体pPICZα-hK1, 该载体转化毕赤酵母(Pichia pastoris)宿主菌X33, 通过提高YPD平板和培养液中抗生素Zeocin的浓度(500~700 μg/mL), 筛选能高水平表达重组人激肽释放酶-1(Recombinant human kallikrein 1, rhK1)的P. pastoris工程菌株。在30 L发酵罐中发酵的表达条件为30oC、pH 6.0, 诱导64 h时, 发酵上清中rhK1产量达到6500 u/L(约1.25 g/L)。表达的rhK1有N-型糖基化程度不同的两种类型, 分别是分子量偏大的rhK1-H和分子量略小的rhK1-L。通过苯基疏水作用、Cu2+螯合以及阴离子交换层析纯化, 从每升发酵上清中可获得0.28 g的rhK1-H和0.62 g的rhK1-L, 纯化的总得率约为72%, 纯度不低于96%。到目前为止, 该研究获得的rhK1产量高于其他研究者的结果。     

Human seminal proteinase and prostate-specific antigen are the same protein

Human seminal proteinase and prostate-specific antigen (PSA) were each isolated from human seminal fluid and compared. Both are glycoproteins of 32-34 kDa with protease activities. Based on some physicochemical,enzymatic and immunological properties,it is concluded that these proteins are in fact identical.The protein exhibits properties similar to kallikrein-like serine protease, trypsin,chymotrypsin and thiol acid protease.Tests of the activity of the enzyme against some potential natural and synthetic substrates showed that bovine serum albumin was more readily hydrolysed than casein.The results of this study should be useful in purifying and assaying this protein.Based on published studies and the present results,the broad proteolytic specificity of human seminal proteinase suggests a role for this protein in several physiological functions.     

Production and activation of recombinant hK2 with propeptide mutations resulting in high expression levels.

Human glandular kallikrein 2 (hK2) is a serine protease expressed mainly by the prostate gland with 80% identity in primary structure to prostate specific antigen (PSA). hK2 has proven to be a useful marker of prostate cancer which can be used in combination with PSA to better discriminate between prostate cancer and benign prostate hyperplasia. The studies on hK2 have been hampered by its very low phyciological levels (6 microgram.mL-1), its close similarity to PSA, and the low expression levels obtained using recombinant procedures to produce hK2 (0.7 mg.L-1). We have now generated propeptide mutations of hK2 which can be used to isolate stable, inactive prohK2 mutants. Compared with wild-type hK2, expression of the propeptide hK2 mutants increases the expression levels up to 15-40-fold giving 10-30 mg hK2.L-1. These results indicate that the low expression levels of wild-type hK2 are related to the activation or autoactivation of the wild-type enzyme and the instability of the active protease in cell culture and possibly also in tissue. The purified mutant hK2 may be activated by either enterokinase or factor Xa to generate an enzyme for use in functional studies with the characteristics of the original wild-type protein. Further, the stable inactive mutant hK2 protein may be used for immunizations to generate novel monoclonal antibodies, used as standard material for clinical assays or in crystallization studies where large quantities of protein are required.     

Isolation and characterization of a basic carboxypeptidase from human seminal plasma

A carboxypeptidase which cleaves the C-terminal arginine or lysine from peptides was purified by a two-step procedure; gel filtration on Sephacryl S-300 and affinity chromatography on arginine-Sepharose. The activity increased 280% after the first step, indicating the removal of an inhibitor from the crude starting material. The activity in the crude seminal plasma eluted from the Sephacryl S-300 column with an apparent Mr 98,000 and after purification with an Mr 67,000, indicating that it binds to another protein in the crude seminal plasma. When analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a single band at Mr 53,000 was seen which was converted to two smaller bands (Mr 32,000 and/or 26,000) after reduction. The seminal plasma carboxypeptidase has a neutral pH optimum, is inhibited by o-phenanthroline and by the inhibitor of carboxypeptidase B-type enzymes, 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, and can be activated by cobalt. The purified enzyme has a high specific activity (67.8 mumol/min/mg) with the ester substrate benzoyl (Bz)-Gly-argininic acid and readily cleaves Bz-Ala-Lys, Bz-Gly-Arg, and Bz-Gly-Lys. It also hydrolyzes biologically active peptides such as bradykinin (Km = 6 microM, kcat = 43 min-1), Arg6-Met5-enkephalin (Km = 103 microM, kcat = 438 min-1), and Lys6-Met5-enkephalin (Km = 848 microM, kcat = 449 min-1). The seminal plasma carboxypeptidase did not cross-react with antiserum to human plasma carboxypeptidase N; other properties distinguish it from the blood plasma enzyme as well as from pancreatic carboxypeptidase B and granular, acid carboxypeptidase H (enkephalin convertase). The carboxypeptidase could be involved in the control of fertility by activating or inactivating peptide hormones in the seminal plasma. In addition it could contribute to the degradation of basic proteins during semen liquefaction.     

Cathepsin B efficiently activates the soluble and the tumor cell receptor-bound form of the proenzyme urokinase-type plasminogen activator (Pro-uPA)

Action of purified human cathepsin B on recombinant single-chain urokinase-type plasminogen activator (pro-uPA) generated enzymatically active two-chain uPA (HMW-uPA), which was indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot from plasmin-generated HMW-uPA and from elastase- or thrombin-generated inactive two-chain urokinase-type plasminogen activator. Preincubation of cathepsin B with E-64 (transepoxysuccinyl-L-leucylamino- (4-guanidino)butane, a potent inhibitor for cathepsin B) prior to the addition of pro-uPA prevented the activation of pro-uPA. The cleavage site within the cathepsin B-treated urokinase-type plasminogen activator (uPA) molecule, determined by N-terminal amino acid sequence analysis, is located between Lys158 and Ile159. Pro-uPA is cleaved by cathepsin B at the same peptide bond that is cleaved by plasmin or kallikrein. Binding of cathepsin B-activated pro-uPA to the uPA receptor on U937 cells did not differ from that of enzymatically inactive pro-uPA, indicating an intact receptor-binding region within the growth factor-like domain of the cathepsin B-treated uPA molecule. Not only soluble but also tumor cell receptor-bound pro-uPA could be efficiently cleaved by cathepsin B to generate enzymatically active two-chain uPA. Thus, cathepsin B can substitute for plasmin in the proteolytic activation of pro-uPA to enzymatically active HMW-uPA. In contrast, no significant activation of pro-uPA by cathepsin D was observed. As tumor cells may produce both pro-uPA and cathepsin B, implications for the activation of tumor cell-derived pro-uPA by cellular proteases may be considered.     

Determination and analysis of antigenic epitopes of prostate specific antigen (PSA) and human glandular kallikrein 2 (hK2) using synthetic peptides and computer modeling.

Prostate specific antigen (PSA) and human glandular kallikrein 2 (hK2), produced essentially by the prostate gland, are 237-amino acid monomeric proteins, with 79% identity in primary structure. Twenty-five anti-PSA monoclonal antibodies (Mabs) were studied for binding to a large array of synthetic linear peptides selected from computer models of PSA and hK2, as well as to biotinylated peptides covering the entire PSA sequence. Sixteen of the Mabs were bound to linear peptides forming four independent binding regions (I-IV). Binding region I was localized to amino acid residues 1-13 (identical sequence for PSA and hK2), II (a and b) was localized to residues 53-64, III (a and b) was localized to residues 80-91 (= kallikrein loop), and IV was localized to residues 151-164. Mabs binding to regions I and IIa were reactive with free PSA, PSA-ACT complex, and with hK2; Mabs binding to regions IIb, IIIa, and IV were reactive with free PSA and PSA-ACT complex, but unreactive with hK2; Mabs binding to region IIIb detected free PSA only. All Mabs tested (n = 7) specific for free PSA reacted with kallikrein loop (binding region IIIb). The presence of Mabs interacting with binding region I did not inhibit the catalytic activity of PSA, whereas Mabs interacting with other binding regions inhibited the catalysis. Theoretical model structures of PSA, hK2, and the PSA-ACT complex were combined with the presented data to suggest an overall orientation of PSA with regard to ACT.     

Activation of the zymogen to urokinase-type plasminogen activator is associated with increased interdomain flexibility

A key regulatory step for serine proteases of the trypsin clan is activation of the initially secreted zymogens, leading to an increase in activity by orders of magnitude. Zymogen activation occurs by cleavage of a single peptide bond near the N-terminus of the catalytic domain. Besides the catalytic domain, most serine proteases have N-terminal A-chains with independently folded domains. Little is known about how zymogen activation affects the interplay between domains. This question is investigated with urokinase-type plasminogen activator (uPA), which has an epidermal growth factor domain and a kringle domain, connected to the catalytic domain by a 15-residue linker. uPA has been implicated under several pathological conditions, and one possibility for pharmacological control is targeting the conversion of the zymogen pro-uPA to active uPA. Therefore, a small-angle X-ray scattering study of the conformations of pro-uPA and uPA in solution was performed. Structural models for the proteins were derived using available atomic-resolution structures for the various domains. Active uPA was found to be flexible with a random conformation of the amino-terminal fragment domain with respect to the serine protease domain. In contrast, pro-uPA was observed to be rigid, with the amino-terminal fragment domain in a fixed position with respect to the serine protease domain. Analytical ultracentrifugation analysis supported the observed difference between pro-uPA and uPA in overall shape and size seen with small-angle X-ray scattering. Upon association of either of two monoclonal Fab (fragment antigen-binding) fragments that are directed against the catalytic domain of, respectively, pro-uPA and uPA, rigid structures were formed.     

Thiophilic-interaction chromatography of enzymatically active tissue prostate-specific antigen (T-PSA) and its modulation by zinc ions

Prostate-specific antigen (PSA) is a serine protease secreted both by normal prostate glandular epithelial cells and prostate cancer cells. We explored "thiophilic-interaction chromatography" (TIC) to isolate tissue prostate-specific antigen (T-PSA) from fresh human prostate cancer tissue harvested by radical prostatectomy for the purpose to characterize T-PSA for its enzymatic activity and sensitivity to zinc ions. We have shown, for the first time, that T-PSA has strong affinity for the thiophilic gel (T-gel). The average recovery of T-PSA from T-gel is over 87%. The presence of PSA in the column eluate was confirmed by ELISA and SDS/PAGE. Western blot developed with monoclonal antibody to PSA revealed that T-PSA was predominantly in the "free" form having a molecular weight of 33 kDa. Furthermore, T-PSA was found to be enzymatically active. T-PSA was found to be less enzymatically active as compared to seminal plasma PSA. The inhibition of enzymatic activity of both f-PSA and T-PSA over a wide range of concentrations of Zn(2+) ions (10nM to 50 microM) was comparable. In contrast, the enzymatic activity of chymotrypsin, another serine-protease, was affected differently. At higher concentrations of Zn(2+) (10 microM and higher) the enzymatic activity of chymotrypsin was inhibited, whereas, at lower concentrations of Zn(2+) (5 microM and lower), the enzymatic activity was enhanced.     

Crystal structure of a prostate kallikrein isolated from stallion seminal plasma: a homologue of human PSA

Prostate-specific kallikrein, a member of the gene family of serine proteases, was initially discovered in semen and is the most useful serum marker for prostate cancer diagnosis and prognosis. We report the crystal structure at 1.42A resolution of horse prostate kallikrein (HPK). This is the first structure of a serine protease purified from seminal plasma. HPK shares extensive sequence homology with human prostate-specific antigen (PSA), including a predicted chymotrypsin-like specificity, as suggested by the presence of a serine residue at position S1 of the specificity pocket. In contrast to other kallikreins, HPK shows a structurally distinct specificity pocket. Its entrance is blocked by the kallikrein loop, suggesting a possible protective or substrate-selective role for this loop. The HPK structure seems to be in an inactivated state and further processing might be required to allow the binding of substrate molecules. Crystal soaking experiments revealed a binding site for Zn(2+) and Hg(2+), two known PSA inhibitors.