Alteration of catalytic properties of chymosin by site-directed mutagenesis.
Artificial mutations of chymosin by recombinant DNA techniques were generated to analyze the structure--function relationship in this characteristic aspartic proteinase. In order to prepare the mutant enzymes in their active form, we established procedures for purification of correctly refolded prochymosin from inclusion bodies produced in Escherichia coli transformants and for its subsequent activation. Mutagenesis by linker insertion into cDNA produced several mutants with an altered ratio of milk clotting activity to proteolytic activity and a different extent of stability. In addition to these mutants, several mutants with a single amino acid exchange were also constructed by site-directed mutagenesis and kinetic parameters of these mutant enzymes were determined by using synthetic hexa- and octa-peptides as substrates. Exchange of Tyr75 on the flap of the enzyme to Phe caused a marked change of substrate specificity due to the change of kcat or Km, depending on the substrate used. Exchange of Val110 and Phe111 also caused a change of kinetic parameters, which indicates functional involvement of these hydrophobic residues in both the catalytic function and substrate binding. The mutant Lys220----Leu showed a marked shift of the optimum pH to the acidic side for hydrolysis of acid-denatured haemoglobin along with a distinct increase in kcat for the octa-peptide in a wide pH range.
Suzuki
J
J
Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, Japan.
Sasaki
K
K
Sasao
Y
Y
Hamu
A
A
Kawasaki
H
H
Nishiyama
M
M
Horinouchi
S
S
Beppu
T
T
D000596:Amino Acids / Q000502:physiology; D001483:Base Sequence; D002384:Catalysis; D012085:Chymosin / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004247:DNA; D004792:Enzyme Precursors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000596:Amino Acids; D004792:Enzyme Precursors; D004247:DNA; C032443:prorennin; D012085:Chymosin