Changing the inhibitory specificity and function of Cucurbita maxima trypsin inhibitor-V by site-directed mutagenesis.
Cucurbita maxima trypsin inhibitor-V (CMTI-V) is also a specific inhibitor of human blood coagulation factor beta-factor XIIa. A recombinant version of CMTI-V has allowed probing of roles of individual amino acid residues including the reactive site residue, lysine (P1), by site-directed mutagenesis. The K44R showed at least a 5-fold increase in inhibitory activity toward human beta-factor XIIa, while there was no change toward bovine trypsin. This result demonstrates that beta-factor-XIIa prefers an arginine residue over lysine residue, while trypsin is non-specific to lysine or arginine in its binding pocket. On the other hand, the specificity of CMTI-V could be changed from trypsin to chymotrypsin inhibition by mutation of the P1 residue to either leucine or methionine (K44L or K44M).
Wen
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L
Department of Chemistry, Western Illinois University, Macomb 61455.
Lee
I
I
Chen
G
G
Huang
J K
JK
Gong
Y
Y
Krishnamoorthi
R
R
D000818:Animals; D001120:Arginine; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism; D015956:Factor XIIa / Q000037:antagonists & inhibitors; D006801:Humans; D008239:Lysine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*
C081870:CMTI protein, Cucurbita maxima; D010940:Plant Proteins; D014361:Trypsin Inhibitors; D001120:Arginine; D002918:Chymotrypsin; D015956:Factor XIIa; D014357:Trypsin; D008239:Lysine