Subtilisin proteins

Bryan PN, Rollence ML, Pantoliano MW, Wood J, Finzel BC, Gilliland GL, Howard AJ, Poulos TL (1986) Proteases of enhanced stability characterization of a thermostable variant of subtilisin. Proteins Structure, Function and Genetics. 1 326-334... 

Animals exposed to 150 pg of subtilisin protein. m for 15 min per day for 5 consecutive days developed immediate-onset and late-onset pulmonary responses when re-exposed to the protein aerosol (Thome et al., 1986). The immediate-onset pulmonary responses were detected as significant increases in the breathing rate. The late-onset responses were also detected as significant increases in breathing rate accompanied by febrile responses. In addition, the animals possessed allergic antibody specific to subtilisin, although not every animal with antibody developed pulmonary symptoms (Hillebrand et al., 1987). Animals exposed to 8 or 41 pg of subtilisin protein, nr3 did not experience pulmonary symptoms, but did develop low levels of antibody to subtilisin. These experiments showed that immune reactivity to this occupational allergen occurred at exposure levels lower than those required for the elicitation of pulmonary responses. 

McPhalen, C. A., James, M. N. G. Structural comparison of two serine proteinase-protein inhibitor complexes Eglin-C-Subtilisin Carlsberg and CI-2-subtilisin novo. Biochemistry 27 (1988) 6582-6598... 

Subtilisins are a group of serine proteinases that are produced by different species of bacilli. These enzymes are of considerable commercial interest because they are added to the detergents in washing powder to facilitate removal of proteinaceous stains. Numerous attempts have therefore recently been made to change by protein engineering such properties of the subtilisin molecule as its thermal stability, pH optimum, and specificity. In fact, in 1988 subtilisin mutants were the subject of the first US patent granted for an engineered protein. 

The active site of subtilisin is outside the carboxy ends of the central p strands analogous to the position of the binding sites in other a/p proteins as discussed in Chapter 4. Details of this active site are surprisingly similar to those of chymotrypsin, in spite of the completely different folds of the two enzymes (Figures 11.14 and 11.9). A catalytic triad is present that comprises residues Asp 32, His 64 and the reactive Ser 221. The negatively charged oxygen atom of the tetrahedral transition state binds in an oxyanion hole,... 

Transition-state stabilization in subtilisin is dissected by protein engineering... 

The subtilisin mutants described here illustrate the power of protein engineering as a tool to allow us to identify the specific roles of side chains in the catalytic mechanisms of enzymes. In Chapter 17 we shall discuss the utility of protein engineering in other contexts, such as design of novel proteins and the elucidation of the energetics of ligand binding to proteins. 

A structural anomaly in subtilisin has functional consequences Transition-state stabilization in subtilisin is dissected by protein engineering Catalysis occurs without a catalytic triad Substrate molecules provide catalytic groups in substrate-assisted catalysis Conclusion Selected readings... 

Furin, also known as paired basic amino-acid-cleaving enzyme (PACE), is a membrane bound subtilisin-like serine protease of the irons Golgi compartment. It is ubiquitously expressed and mediates processing of many protein precursors at Arg-X-Lys/Arg-Arg sites. 

The family of serine proteases has been subjected to intensive studies of site-directed mutagenesis. These experiments provide unique information about the contributions of individual amino acids to kcat and KM. Some of the clearest conclusions have emerged from studies in subtilisin (Ref. 9), where the oxyanion intermediate is stabilized by t>e main-chain hydrogen bond of Ser 221 and an hydrogen bond from Asn 155 (Ref. 2). Replacement of Asn 155 (e.g., the Asn 155— Ala 155 described in Fig. 7.9) allows for a quantitative assessment of the effect of the protein dipoles on Ag. ... 

Enzyme active sites, 136,148, 225. See also Protein active sites in carbonic anhydrase, 197-199 in chymotrypsin, 173 in lysozyme, 153, 157 nonpolar (hypothetical site), 211-214 SNase, 189-190,190 steric forces in, 155-158, 209-211, 225 in subtilisin, 173 viewed as super solvents, 227 Enzyme cofactors calcium ... 

The first choice of enzyme to add to a detergent is practically always a protease. The proteases in modem detergents are subtilisins which are microbial enzymes from Bacillus. The subtilisins consist of approximately 270 amino acids and are heart-shaped molecules with a binding cleft and a binding pocket to which substrates such as protein stains can be bound by non-covalent forces. 

A large number of potential reversible protease inhibitors exist (Laskowski Kato, 1980). Protein protease inhibitors like Strepromyces Subtilisin Inhibitor (SSI) (Hiromi et al, 1985) and Chymotrypsin Inhibitor (CI-2) (Jonassen, 1980 and McPhalen James, 1988) are known to be very strong inhibitors with inhibition constants at or below 10"10 M. 

Hiromi, K Akasaka, K Mitsui, Y Tonomura, B Murao, S (eds) (1985) Protein Protease Inhibitor - The Case of Streptomyces Subtilisin Inhibitor. Elsevier Amsterdam - Oxford - New York. 

A second approach that can be adopted to overcome the intrinsic requirement for cysteine at the N-terminus of C-terminal fragment utilizes the enzyme subtiligase, a double mutant of subtilisin, which is able to join two unprotected peptides. Thioester-modified proteins were shown to present good substrates of subtiligase [65]. However, although this approach could be potentially useful for general isotope labeling, the efficiency of this process remains to be proven. 

For single-tryptophan proteins there is some correlation between blue-shifted fluorescence emission maximum and phosphorescence lifetime (Table 3.2). Another correlation is that three of the proteins which exhibit phosphorescence, azurin, protease (subtilisin Carlsberg), and ribonuclease Tlt are reported to show resolved fluorescence emission at 77 K. Both blue-shifted emission spectra and resolved spectra are characteristic of indole in a hydrocarbon-like matrix. 

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