Conversion of trypsin to a functional threonine protease

Abstract

The hydroxyl group of a serine residue at position 195 acts as a nucleophile in the catalytic mechanism of the serine proteases. However, the chemically similar residue, threonine, is rarely used in similar functional context. Our structural modeling suggests that the Ser 195 --> Thr trypsin variant is inactive due to negative steric interaction between the methyl group on the beta-carbon of Thr 195 and the disulfide bridge formed by cysteines 42 and 58. By simultaneously truncating residues 42 and 58 and substituting Ser 195 with threonine, we have successfully converted the classic serine protease trypsin to a functional threonine protease. Substitution of residue 42 with alanine and residue 58 with alanine or valine in the presence of threonine 195 results in trypsin variants that are 10(2) -10(4) -fold less active than wild type in kcat/KM but >10(6)-fold more active than the Ser 195 --> Thr single variant. The substitutions do not alter the substrate specificity of the enzyme in the P1'- P4' positions. Removal of the disulfide bridge decreases the overall thermostability of the enzyme, but it is partially rescued by the presence of threonine at position 195.

Figure 1.

(A) Accepted mechanism of serine proteases. Schematic of the catalytic triad in a serine protease in the presence of a bound Arg-containing substrate. The hydroxyl group of Ser 195 attacks the carbonyl of the Arg residue. The hydrogen atom of the β-carbon closest to the S1′ pocket is in bold. (B) Arrangement of the catalytic triad in wild-type trypsin. The arrangement of the residues in the active site of wild-type rat trypsin are shown in stick model. The amino acid side chains of interest are in green for wild type and cyan for the S195T variant. Oxygen atoms are red, nitrogen atoms are blue, and sulfur atoms are orange. This figure was constructed with PYMOL using the coordinates from PDB file 1ANE. (C) Model of optimal arrangement of the catalytic triad in C42A/C58V/S195T trypsin (AVT-Tn) variant. Ala 42,Val 58, and Thr 195 are labeled. The expected arrangement for an active threonine protease variant of trypsin. The hydroxyl group of Thr 195 is in a position comparable to that of Ser 195 in the native enzyme.

Figure 2.

Activity of trypsin variants. The dark bars are activities of the trypsin variants toward Z-GPR-SBzl. The light bars are activities toward Z-GPR-pNA (pNA). The striped bars are activities toward Z-GPR-AMC (AMC). The abbreviations for the trypsin variants are as follows: WT = wild type; S195T = Ser 195 → Thr; AA-Tn = C42A/C58A trypsin; AV-Tn = C42A/C58V trypsin; AAT = C42A/C58A/S195T trypsin; AVT-Tn = C42A/C58V/S195T trypsin. (This result differs slightly from those of previous experiments in which the S195T variant was reported to have activity [Corey and Craik 1992]. This difference may be resolved by considering differences in experimental design. In the previous study, enzyme and substrate assay mixtures were incubated at 25°C until at least one turnover occurred. In the case of the S195T, an assay time >8 h was required to observe one turnover. Results obtained from such a limited data set over such a large time scale are more likely to be susceptible to error. To resolve any potential conflicts, identical experiments with S195T Tn were carried out in parallel for this study.)

Figure 3.

Substrate specificity profiles. The amino acid preferences for substrate positions P1–P4 were determined for each of the variants and wild-type trypsin as described in Materials and Methods. Panels A–D correspond to preferences for positions P1–P4, respectively, for wild-type trypsin. Panels E–H correspond to preferences for positions P1–P4, respectively, for the AVT-Tn triple variant. The AA-Tn, AV-Tn, and AAT-Tn variants yielded similar results.

Figure 4.

Temperature dependence of hydrolytic activity. The thermostability of AVT-Tn and AV-Tn variants was determined by a modified kinetic assay as described in Materials and Methods. The rate of Z-GPR-pNA hydrolysis catalyzed by WT at 37°C (♦), C42A/C58V/S195T at 25°C (•) and 37°C (▪), and C42A/C58V at 25°C (○) and 37°C (□).

Scheme 1.

Mechanism of serine proteases. E is the free enzyme, ES is the enzyme–substrate complex, EA is the covalent acyl enzyme intermediate, P1 and P2 are the N- and C-terminal hydrolysis products, respectively.