Mutation in trypsin

The Asp 189-Lys mutation in trypsin causes unexpected changes in substrate specificity... 

Warshel and collaborators (Warshel and Sussman, 1986 Warshel et al., 1988) developed the empirical valence bond method for obtaining free-energy differences and activation free energies. The effects of Gly-to-Ala mutations in trypsin were accurately simulated. This method was earlier applied to calculation of the potential surface for general acid catalysis of a disaccharide in solution and bound to lysozyme (Warshel and Weiss, 1980). 

The deep hydrophobic SI pockets are very similar in all three proteins. All residues are conserved except for an A190S mutation in trypsin vhich makes its pocket smaller and slightly more hydrophilic. While this is reflected by the pseudo-contour fields, the authors doubt that this difference can be exploited to design selective ligands. 

A, MeloandM. J. Ramos,/. Peptide Res., SO, 382 (1997). The Nature of Trypsin-Pancreatic Trypsin Inhibitor Binding Free Energy Calculation of Tyr39 -> Phe39 Mutation in Trypsin,... 

Mutations in the specificity pocket of trypsin, designed to change the substrate preference of the enzyme, also have drastic effects on the catalytic rate. These mutants demonstrate that the substrate specificity of an enzyme and its catalytic rate enhancement are tightly linked to each other because both are affected by the difference in binding strength between the transition state of the substrate and its normal state. 

Chobert, J.-M., Briand, L., and Haertle, T. 1998b. Influence of G187W/K188F/D189Y mutation in the substrate-binding pocket of trypsin on (3-casein processing. J. Food Biochem. 22, 529-545. 

Castro MJ, Anderson S. Alanine point-mutations in die reactive region of bovine pancreatic trypsin inhibitor effects on the kinetics and thermodynamics of binding to beta-trypsin and alpha-chymotrypsin. Biochemistry 1996 35 11435-11446. 

The contribution of Asp to maintaining His in such an orientation that can accept a proton from Ser is considered to be of minor importance since the electrostatic contribution to the difference in free energy between native and mutant serine proteinases accounts for most of the calculated effects of the mutations (Asp to Asn in trypsin, Asp to Ala in subtilisin). The other stabilizing effect is of the oxyanion hole on the negatively charged TI, which is also electrostatic in nature. 

Hirota, M. et ah. Significance of trypsin inhibitor gene mutation in the predisposition to pancreatitis, Int. 

The results of experiments in which the mutation was made were, however, a complete surprise. The Asp 189-Lys mutant was totally inactive with both Asp and Glu substrates. It was, as expected, also inactive toward Lys and Arg substrates. The mutant was, however, catalytically active with Phe and Tyr substrates, with the same low turnover number as wild-type trypsin. On the other hand, it showed a more than 5000-fold increase in kcat/f m with Leu substrates over wild type. The three-dimensional structure of this interesting mutant has not yet been determined, but the structure of a related mutant Asp 189-His shows the histidine side chain in an unexpected position, buried inside the protein. 

As these experiments with engineered mutants of trypsin prove, we still have far too little knowledge of the functional effects of single point mutations to be able to make accurate and comprehensive predictions of the properties of a point-mutant enzyme, even in the case of such well-characterized enzymes as the serine proteinases. Predictions of the properties of mutations using computer modeling are not infallible. Once produced, the mutant enzymes often exhibit properties that are entirely surprising, but they may be correspondingly informative. 

JOFUKU, K.D., SCHIPPER, R.D., GOLDBERG, R.B., A ffameshift mutation prevents Kunitz trypsin-inhibitor messenger-RNA accumulation in soybean embryos, Plant Cell, 1989,1,427-435. 

The trypsinized cultures are counted and a sample is assessed for survival as for the cytotoxicity assay. In addition, an appropriate number of cells are reseeded for estimation of mutation frequency at the day 8 expression time. The cells are transferred to roller bottles (usually 490 cm2) for this stage. The bottles are gassed with pure CO2, the tops are tightened and the bottles are incubated at 37°C on a roller machine (approximate speed 0.5-1.0 rev min-1). Usually 106 7 8 9 viable cells are reseeded in 50 ml of Eagle s medium containing serum, but more cells are required at the toxic dose levels. 

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