Mutagenesis, biochemical characterization and X-ray structural analysis of point mutants of bovine chymosin

Mark G. Williams, Julie Wilsher, Phil Nugent, Alan Mills, V. Dhanaraj, Milan Fabry, Juraj Sedlácek, J. M. Uusitalo, M. E. Penttila, Jim E. Pitts, Tom L. Blundell

    Research output: Contribution to journalArticleScientificpeer-review

    17 Citations (Scopus)

    Abstract

    Chymosin B point mutants, A115T and G243D (chymosin A), were expressed in Escherichia coli and Trichoderma reesei respectively, characterized biochemically, crystallized and studied by X-ray analysis at 2.3 and 2.8 Å resolutions respectively. The three-dimensional structures showed that the mutations gave rise to local conformational changes only when compared with that of chymosin B. Kinetic analysis of the A115T mutant with a six residue synthetic peptide revealed a reduction in K(m) with respect to the wild type, possibly caused by the small local changes in the vicinity of S1 and S3. Although, kinetic analyses of the G243D mutant using the short substrate showed reduced catalytic activity, use of a 15 residue substrate based on residues 98-112 of κ-casein, the natural substrate, revealed an increase in the k(cat) compared with chymosin B, probably a consequence of the charge introduced that may interact with the substrate between P4 and P8.

    Original languageEnglish
    Pages (from-to)991-997
    Number of pages7
    JournalProtein Engineering
    Volume10
    Issue number9
    Publication statusPublished - 1 Dec 1997
    MoE publication typeA1 Journal article-refereed

    Keywords

    • Aspartic proteinase
    • Chymosin
    • Crystal structure
    • Mutant
    • Substrate

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