Purification and characterization of S1 mutants of alpha-lytic protease having altered catalytic properties.
A procedure is described for purifying alpha-lytic protease and its mutants from culture supernatants of recombinant Escherichia coli. The method affords substantial amounts (approx. 80 mg) of homogeneous enzyme. We compared the cleavage preferences of wild-type alpha-lytic protease and of mutants containing the substitutions Ala190 ("parent"), Ala190/Val192/His213/Met218 (mutant 1), Ala190/His213/Leu218 (mutant 9), and Ala190/Thr213/Leu218 (mutant 55), and for each enzyme we found broad agreement between the results obtained with synthetic ester and amide substrates. Kinetic constants were determined for the purified enzymes using selected tetrapeptide p-nitroanilide substrates. Mutant 55 had broad specificity and high activity. In terms of kcat/Km it cleaved at Met and Phe residues two to three times as effectively as the Ala190 enzyme and cleaved at Ala 7 times more effectively than the wild-type protease. The Ala190/His213 enzymes showed a preference for cleavage at His and Met residues. Not only were their kcat values for cleavage at His increased (in relation to the Ala190 enzyme) by an order of magnitude, but they also exhibited large decreases in kcat/Km for cleavage at other residues; for example, the value for cleavage at Phe was 400- to 600-fold lower. Mutant 9 cleaved a recombinant IGF-II fusion protein at a unique His residue and also at a nearby Asn residue.
Haggett
K D
KD
Sydney Laboratory, CSIRO Division of Biomolecular Engineering, North Ryde, NSW, Australia.
Graham
L D
LD
Milner
S J
SJ
Whittaker
R G
RG
D000577:Amides / Q000378:metabolism; D000595:Amino Acid Sequence; D002384:Catalysis; D004795:Enzyme Stability; D004926:Escherichia coli; D004952:Esters / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000577:Amides; D004952:Esters; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C021826:myxobacter alpha-lytic proteinase