Trypsin mutation

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

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. 

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. 

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. 

The primary goal of peptide mapping is the verification of the amino acid sequence deduced from the genetic code of the recombinant protein. The protein backbone gets cleaved by typically two or three different endoproteinases like Lys-C, trypsin, and Glu-C to achieve maps with sequence-overlapping peptide fragments. These peptide mixtures can then be separated by LC or CE and analyzed on-line by MS to obtain sequence information. Often simple mass analysis matches the predicted primary sequence of the protein. However, sometimes mutations can lead to isobaric masses of peptides that can be overseen, if no further sequence analysis like N-terminal Edman sequencing and MS/MS is carried out. 

The /8 subunit of tryptophan synthase from E. coli is inactivated by a mutation at Gly-281 (G281R).108 Gly-281 is located at a sharp turn in the trypsin-sensitive, subdomain (residues 260-310) that makes several contacts with the a subunit.7) The G281R mutation alters the catalytic properties of the isolated /8 subunit and weakens association with the a subunit. The mutation may interfere with hydrophobic interactions between the N-terminal and C-terminal domains of the /8 subunit and prevent a conformational change that affects catalytic properties and subunit interaction.108 Insertion of arginine or tryptophan between tyrosine 279 and phenylalanine 280 of the /3 subunit greatly weakens subunit interaction and decreases catalytic activity (X.-J. Yang and E.W. Miles, unpublished results). 

The same lysyl residue 188 was replaced with histidine in order to build a metal chelation site in the substrate-binding pocket of trypsin (Briand et al., 1997). K188H mutation did not affect catalytic efficiency at all. In the presence of Cu2+, trypsin K188H exhibited a 30- to 100-fold increase of Km, while kcat was only slightly decreased (Table VII). Hydrolytic activity of this mutant could be fully restored by addition of EDTA. Thus, in contrast to the chelation of the active site, a different mode of metal-dependent inhibition of the activity of trypsin by building a co-ordination site in the substrate-binding pocket of the protease was achieved. 

Synthetic substrates allow rapid determination of the catalytic constants of an enzyme. Nevertheless, it is known that the environment of the peptide bond depends largely on physico-chemical conditions of the applied media, and imposed steric hindrance. Since these parameters are important, the hydrolysis of purified (3-casein was studied at different pHs. The kinetic analysis revealed that the mutant conserved the native trypsin capacity to hydrolyze peptide bonds containing arginyl and lysyl residues. The optimal pH of activity changed considerably according to the mutation. 

The use of (3-casein as a test substrate presents, besides the importance of this protein in the food industry, advantages of releasing the hydrolysis from several structural limitations characteristic of many other potential native protein substrates. The use of this protein enabled a better understanding of the scope and validity of the results obtained with synthetic substrates. Additionally, the harnessing of mutated trypsins into the processing of (3-casein diversified the peptide products obtained. Most of the observed new cleavage sites were located in the hydrophobic portion of the protein. 

Modifications introduced by the mutations were central to the alteration of the specificities of the enzymes studied, which were capable of cleaving (3-casein at many new sites, for example, hydrolyzing the fragment Argl-Lysl05, reported to be a trypsin inhibitor (Bouhallab et al, 1997). Since many tryptic inhibitors contain amidated Glu and Asp, and form amyloid structures, the mutants of this type could be used for the hydrolysis of the lytically resistant protein structures. 

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. 

Noone PG, Zhou Z, Silverman LM, Jowell PS, Knowles MR, Cohn JA (2001) Cystic fibrosis gene mutations and pancreatitis risk relation to epithelial ion transport and trypsin inhibitor gene mutations. Gastroenterology 121 1310-9... 

Trypsin digests of both wild type HRV virus and the mutant were analyzed using MALDI-TOF and MALDI Fourier transform mass spectrometry (FTMS). For HRV, the mass spectra for both wild-type and mutant were identical except for one peptide occurring at mlz 4700. This corresponds to residues 187-227 in the wild type sequence. The corresponding peak in the mutant mass spectrum occurs at 4783.5 (Fig. 4, inset). This mass difference of 83 Da corresponds exactly to a mutation of a Cys to Trp residue and there are no other possible mutations that would be separated by 83 Da. Since there is only one Cys in the peptide 187-227 at position 199, the mutant can be localized as HRV14-Cysl99Trp, which contains a Trp at position 199 instead of Cys in the wild type. 

SI pockets [22], The three major activities, the peptidylglutamil-hydrolyzing (PGPH), trypsin-like and chymotrypsin-like activities, have been assigned to the three active subunits of ySl, yS2 and yS5, respectively, based on mutational and crystal structure analyses. Furthermore, it has been claimed, based on studies with model peptides and inactivation by inhibitors, that mammalian proteasomes also contain two other peptidase activities, referred to as branched-chain amino acid-preferring and small neutral amino acid-preferring (SNAAP). 

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