The assignment of the reactive sites of the double-headed arrowhead proteinase inhibitor A and B.
The arrowhead proteinase inhibitor A and B (APIA and APIB) are double-headed and multifunctional. Both their primary structure and cDNA sequence have been elucidated. To locate the possible reactive site residues Lys(44), Arg(76) and Arg(87) of APIB predicted according to the sequence comparison with other proteinase inhibitors, the above residues were substituted with Pro by site-directed mutagenesis respectively, and the mutated genes were expressed in the yeast secretion system. The mutant K(44)P-APIB displayed the same inhibitory activity as APIB does, while the mutants R(76)P-APIB and R(87)P-APIB could only inhibit one molecule, instead of two molecules of trypsin, indicating that Arg(76) and Arg87 but not Lys(44) are the two reactive sites of APIB. In order to further confirm this result, more mutants of APIB (K(44)P-R(76)P-APIB, K(44)P-R(87)P-APIB,  R(76)P-R(87)P-APIB) were designed, in each mutant only one of the three possible reactive sites remained unchanged. Both the mutants K(44)P-R(76)P-APIB and K(44)P-R(87)P-APIB could only inhibit one molecule of trypsin, while R(76)P-R(87)P-APIB could no longer inhibit trypsin intensively, thus the residues Arg(76) and Arg(87) are definitely the reactive sites of APIB. Their K(i) were measured to be 0.39 nmol/L and 0.47 nmol/L, respectively. The mutant R(87)L-APIB lost about half activity towards trypsin but could inhibit one molecule of chymotrypsin as the wide type APIA did, indicating that Leu(87) was the reactive site towards trypsin but could inhibit one molecule of APIA for inhibiting chymotrypsin.
Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031, China. chi@sunm.shcnc.ac.cn
Jiong
J
Li
Kang-Cheng
KC
Ruan
Cheng-Wu
CW
Chi
D000595:Amino Acid Sequence; D001665:Binding Sites; D002874:Chromosome Mapping; D002918:Chymotrypsin / Q000037:antagonists & inhibitors; D029624:Ferns; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010940:Plant Proteins / Q000235:genetics*; D015842:Serine Proteinase Inhibitors / Q000235:genetics*; D014361:Trypsin Inhibitors
C080655:API-A protein, Sagittaria sagittifolia; D010940:Plant Proteins; D015842:Serine Proteinase Inhibitors; D014361:Trypsin Inhibitors; C047493:proteinase inhibitor B, arrowhead root; D002918:Chymotrypsin