Gary S. Coombs

Substrate specificity of prostate-specific antigen (PSA)

BACKGROUND The serine protease prostate-specific antigen (PSA) is a useful clinical marker for prostatic malignancy. PSA is a member of the kallikrein subgroup of the (chymo)trypsin serine protease family, but differs from the prototypical member of this subgroup, tissue kallikrein, in possessing a specificity more similar to that of chymotrypsin than trypsin. We report the use of two strategies, substrate phage display and iterative optimization of natural cleavage sites, to identify labile sequences for PSA cleavage. RESULTS Iterative optimization and substrate phage display converged on the amino-acid sequence SS(Y/F)Y decreases S(G/S) as preferred subsite occupancy for PSA. These sequences were cleaved by PSA with catalytic efficiencies as high as 2200-3100 M-1 s-1, compared with values of 2-46 M-1 s-1 for peptides containing likely physiological target sequences of PSA from the protein semenogelin. Substrate residues that bind to secondary (non-S1) subsites have a critical role in defining labile substrates and can even cause otherwise disfavored amino acids to bind in the primary specificity (S1) pocket. CONCLUSION The importance of secondary subsites in defining both the specificity and efficiency of cleavage suggests that substrate recognition by PSA is mediated by an extended binding site. Elucidation of preferred subsite occupancy allowed refinement of the structural model of PSA and should facilitate the development of more sensitive activity-based assays and the design of potent inhibitors.

Distinguishing the Specificities of Closely Related Proteases ROLE OF P3 IN SUBSTRATE AND INHIBITOR DISCRIMINATION BETWEEN TISSUE-TYPE PLASMINOGEN ACTIVATOR AND UROKINASE

Elucidating subtle specificity differences between closely related enzymes is a fundamental challenge for both enzymology and drug design. We have addressed this issue for two intimately related serine proteases, tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), by modifying the technique of substrate phage display to create substrate subtraction libraries. Characterization of individual members of the substrate subtraction library accomplished the rapid, direct identification of small, highly selective substrates for t-PA. Comparison of the amino acid sequences of these selective substrates with the consensus sequence for optimal substrates for t-PA, derived using standard substrate phage display protocols, suggested that the P3 and P4 residues are the primary determinants of the ability of a substrate to discriminate between t-PA and u-PA. Mutagenesis of the P3 and P4 residues of plasminogen activator inhibitor type 1, the primary physiological inhibitor of both t-PA and u-PA, confirmed this prediction and indicated a predominant role for the P3 residue. Appropriate replacement of both the P3 and P4 residues enhanced the t-PA specificity of plasminogen activator inhibitor type 1 by a factor of 600, and mutation of the P3 residue alone increased this selectivity by a factor of 170. These results demonstrate that the combination of substrate phage display and substrate subtraction methods can be used to discover specificity differences between very closely related enzymes and that this information can be utilized to create highly selective inhibitors.

ToIC and DsbA are needed for the secretion of STB, a heat‐stable enterotoxin of Escherichia coli

STB secretion‐deficient mutants were isolated using the synthetic transposon TnβIaM. Cultures were plated using a double‐membrane system of cellulose acetate and nitrocellulose placed on Luria agar plates containing carbenicillin. The STB bound to the underlying nitrocellulose membrane was detected with anti‐STB antibodies. The altered genes of two STB secretion‐deficient mutants were identified by conjugation and complementation as toIC and dsbA. In cultures of well‐characterized dsbA and toIC mutants, STB was absent from the culture supernatant. The role of ToIC and DsbA in the secretion of peptides is discussed.

Crystals of urokinase type plasminogen activator complexes reveal the binding mode of peptidomimetic inhibitors

Urokinase type plasminogen activator (uPA), a trypsin-like serine proteinase, plays an important role in normal tissue re-modelling, cell adhesion, and cell motility. In addition, studies utilizing normal animals and potent, selective uPA inhibitors or genetically modified mice that lack functional uPA genes have demonstrated that uPA can significantly enhance tumor initiation, growth, progression and metastasis, strongly suggesting that this enzyme may be a promising anti-cancer target. We have investigated the structure-activity relationship (SAR) of peptidomimetic inhibitors of uPA and solved high resolution X-ray structures of key, lead small molecule inhibitors (e.g. phenethylsulfonamidino(P4)-D-seryl(P3)-L-alanyl(P2)-L-argininal(P1) and derivatives thereof) in complex with the uPA proteinase domain. These potent inhibitors are highly selective for uPA. The non-natural D-seryl residue present at the P3 position in these inhibitors contributes substantially to both potency and selectivity because, due to its D-configuration, its side-chain binds in the S4 pocket to interact with the uPA unique residues Leu97b and His99. Additional potency and selectivity can be achieved by optimizing the inhibitor P4 residue to bind a pocket, known as S1sub or S1beta, that is adjacent to the primary specificity pocket of uPA.

Kinetic characterization of a peptide inhibitor of trypsin isolated from a synthetic peptide combinatorial library

Abstract Recently Eichler and Houghten 1 have described the use of a combinatorial peptide library to optimize small trypsin inhibitors. Our kinetic analysis of the most inhibitory peptide, YYGAKIYRPDKM, reveals that it is an inefficiently cleaved substrate for trypsin due to a low k cat . possesses a relatively low K M , and may exhibit nonproductive binding.

Positive and negative selectivity in protease evolution: Investigation of the specificities of plasmin, t-PA, u-PA, and PSA using substrate phage display

Plasmin has significantly different preferences for subsite occupancy when compared to t-PA and u-PA. There is a >700,000 fold difference between the best and worst peptide substrates for plasmin, although both contain a P1 arginine. This differential is generated by a combination of interactions at the P4, P3, P2, P1′ and P2′ subsites. Thus it appears that much of the evolutionary pressure guiding the development of plasmin specificity has been directed towards preventing self-cleavage of the plasminogen activation sequence. This negative selectivity contrasts sharply with the positive selectivity that characterizes enzyme recognition in general, but can be rationalized by the need for plasmin to avoid activating its own zymogen and thus short circuiting regulation by t-PA or u-PA.

Site-Directed Mutagenesis and Protein Engineering

Publisher Summary This chapter discusses site-directed mutagenesis and genetic engineering. Protein engineering is defined as the rational manipulation of protein chemistry to elicit novel functional and structural properties. Mutagenesis by exposure to chemical mutagens or ultraviolet light has long been an important tool in genetics. One method of selection for the mutagenic strand involves the use of multiple mutagenic primers in a single in vitro synthesis reaction. One primer contains the desired mutation to be made in the gene of interest, and another primer contains a mutation that repairs a mutant antibiotic resistance gene. This method allows for strong selection against the template strand and provides a simple initial screen for potential mutant clones. Polymerase chain reaction is the basis for several methods for mutagenesis. The ability to amplify a segment of DNA from essentially any template virtually eliminates the need for selection or screening and greatly simplifies troubleshooting if problems occur. It is found that the combination of alanine scanning, deletion mutagenesis, specific site-directed substitutions, and repeated rounds of phage display screening demonstrated that the techniques of protein engineering can be combined to optimize macromolecular function.