Quantitative assessment of enzyme specificity in vivo : P ₂ recognition by Kex2 protease defined in a genetic system

Abstract

The specificity of the yeast proproteinprocessing Kex2 protease was examined in vivo by using a sensitive, quantitative assay. A truncated prepro-␣-factor gene encoding an ␣-factor precursor with a single ␣-factor repeat was constructed with restriction sites for cassette mutagenesis f lanking the single Kex2 cleavage site (-SLDKR2EAEA-). All of the 19 substitutions for the Lys (P 2 ) residue in the cleavage site were made. The wild-type and mutant precursors were expressed in a yeast strain lacking the chromosomal genes encoding Kex2 and prepro-␣-factor. Cleavage of the 20 sites by Kex2, expressed at the wild-type level, was assessed by using a quantitative-mating assay with an effective range greater than six orders of magnitude. All substitutions for Lys at P 2 decreased mating, from 2-fold for Arg to >10 6 -fold for Trp. Eviction of the Kex2-encoding plasmid indicated that cleavage of mutant sites by other cellular proteases was not a complicating factor. Mating efficiencies of strains expressing the mutant precursors correlated well with the specificity (k cat ͞K M ) of purified Kex2 for comparable model peptide substrates, validating the in vivo approach as a quantitative method. The results support the conclusion that K M , which is heavily inf luenced by the nature of the P 2 residue, is a major determinant of cleavage efficiency in vivo. P 2 preference followed the rank order: Lys > Arg >

Functional Properties of Glycosylated Lysozyme Secreted inPichia pastoris

“…This indicates that the signal cleavage enzyme in yeast cleaved another upstream site in addition to the correct signal cleavage site. Bevan et al 18) have reported that the speciˆcity (kcat W KM) of kex2, a yeast proprotein-processing protease, was heavily in‰uenced by the nature of the lysine residue. Therefore, two types of lysozyme were secreted in P. pastoris, as shown in Fig.…”

The requirement for the highly conserved G−1 residue of Saccharomyces cerevisiae tRNAHis can be circumvented by overexpression of tRNAHis and its synthetase

“…Since the lack of mature a-factor in kex2 mutants causes a 10 6 -fold reduction in the frequency of mating (Bevan et al 1998), suppression of a kex2 nonsense mutant provides a convenient and highly sensitive assay to measure nonsense suppression. Moreover, since Kex2 is a serine protease, the required catalytic triad residues (H213, S385, and D175) are each crucial for activity (Fuller et al 1989), and therefore suppression of a kex2-H213am nonsense mutant requires histidine insertion (Fig.…”

“…To ensure histidine-specific nonsense suppression, we used Kex2, a serine protease required for yeast mating (Leibowitz and Wickner 1976;Bevan et al 1998). Since the lack of mature a-factor in kex2 mutants causes a 10 6 -fold reduction in the frequency of mating (Bevan et al 1998), suppression of a kex2 nonsense mutant provides a convenient and highly sensitive assay to measure nonsense suppression.…”

Reduced Proteolysis of Secreted Gelatin and Yps1-Mediated α-Factor Leader Processing in a Pichia pastoris kex2 Disruptant

“…The banding pattern of low-level degradation products in the kex2 strain was not significantly different from that in the wild type. We previously suggested that these minor fragments might have originated from limited cleavage by Kex2 at Arg residues within a suboptimal sequence context because a tailored gelatin with no Arg residues at all was secreted completely intact (47) and because even the relatively discriminative S 2 and S 4 subsites (Schechter and Berger [42] nomenclature) of S. cerevisiae Kex2 have some leeway in the substrate residues that they can accommodate (8,43). Apparently, however, few sites in Col1a1-2 other than Met-Gly-Pro-Arg meet the Kex2 protease's overall substrate requirements, as most of the minor bands were also present in the kex2 disruptant.…”