Protease substrate specificities

Salisbury CM, Maly DJ, Ellman JA (2002) Peptide microarrays for the determination of protease substrate specificity. J Am Chem Soc 124 14868-14870... 

St. Hilaire PM, Willert M, Juliano MA, Juliano L, Meldal M, Fluorescence-quenched solid phase combinatorial libraries in the characterization of cysteine protease substrate specificity, J. Comb. Chem., 1 509-523, 1999. 

Engineering Substrate Specificity. Although the serine proteases use a common catalytic mechanism, the enzymes have a wide variety of substrate specificities. For example, the natural variant subtiHsins of B. amyloliquefaciens (subtiHsin BPN J and B. licheniformis (subtiHsin Carlsberg) possess very similar stmctures and sequences where 86 of 275 amino acids are identical, but have different catalytic efficiencies, toward tetraamino acid -nitroanilide substrates (67). 

Mammals, fungi, and higher plants produce a family of proteolytic enzymes known as aspartic proteases. These enzymes are active at acidic (or sometimes neutral) pH, and each possesses two aspartic acid residues at the active site. Aspartic proteases carry out a variety of functions (Table 16.3), including digestion pepsin and ehymosin), lysosomal protein degradation eathepsin D and E), and regulation of blood pressure renin is an aspartic protease involved in the production of an otensin, a hormone that stimulates smooth muscle contraction and reduces excretion of salts and fluid). The aspartic proteases display a variety of substrate specificities, but normally they are most active in the cleavage of peptide bonds between two hydrophobic amino acid residues. The preferred substrates of pepsin, for example, contain aromatic residues on both sides of the peptide bond to be cleaved. 

Rhinoviruses, which represent the single major cause of common cold, belong to the family of picornavimses that harbors many medically relevant pathogens. Inhibitors of the 3C protease, a cysteine protease, have shown good antiviral potential. Several classes of compounds were designed based on the known substrate specificity of the enzyme. Mechanism-based, irreversible Michael-acceptors were shown to be both potent inhibitors of the purified enzyme and to have antiviral activity in infected cells. 

The matrix metalloendoproteinases (MMPs or matrixins) are a family of zinc and calcium dependent extracellular proteases that collectively degrade most of the protein constituents of the extracellular matrix [9]. There are at least 23 members of this family and are divided primarily on the basis of sequence homology and substrate specificity into the following grouping collagenases (MMP-1, -8, -13, -18) gelatinases... 

The i-poly(3HB) depolymerase of R. rubrum is the only i-poly(3HB) depolymerase that has been purified [174]. The enzyme consists of one polypeptide of 30-32 kDa and has a pH and temperature optimum of pH 9 and 55 °C, respectively. A specific activity of 4 mmol released 3-hydroxybutyrate/min x mg protein was determined (at 45 °C). The purified enzyme was inactive with denatured poly(3HB) and had no lipase-, protease-, or esterase activity with p-nitro-phenyl fatty acid esters (2-8 carbon atoms). Native poly(3HO) granules were not hydrolyzed by i-poly(3HB) depolymerase, indicating a high substrate specificity similar to extracellular poly(3HB) depolymerases. Recently, the DNA sequence of the i-poly(3HB) depolymerase of R. eutropha was published (AB07612). Surprisingly, the DNA-deduced amino acid sequence (47.3 kDa) did not contain a lipase box fingerprint. A more detailed investigation of the structure and function of bacterial i-poly(HA) depolymerases will be necessary in future. 

Peeona, j. j. and Ceaik, C. S. Structural basis of substrate specificity in the serine proteases. Protein Sci 1995, 4, 337-360. 

Another approach for producing proteases with specific properties which differs from the approach based upon mutagenesis of existing proteases is to synthesize peptides which contain the necessary catalytic and substrate binding groups necess for a particular hydrolytic reaction. One can envision rather unlimited... 

Selected entries from Methods in Enzymology [vol, page(s)] Active site, structure, 241, 214 catalytic mechanism, 241, 223-224 crystal structure, 241, 214, 216 comparative studies with HIV protease [catalytic properties, 241, 205-224 evolutionary relationships, 241, 196-197 screening for HIV-1 protease inhibitors, 241, 318-321 structure, 241, 254-257, 280 substrate specificity, 241, 255, 283]. 

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