The
Assignment of the Reactive Sites of the Double-headed
Arrowhead Proteinase Inhibitor A and B
LI Jiong, RUAN Kang-Cheng, CHI Cheng-Wu*
( Laboratory of Proteomics, Institute of Biochemistry and Cell Biology,
Shanghai Institutes for Biological Sciences,
the Chinese Academy of Sciences, Shanghai 200031, China )
Abstract 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 Lys44, Arg76
and Arg87 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 K44P-APIB displayed the
same inhibitory activity as APIB does, while the mutants R76P-APIB
and R87P-APIB could only inhibit one molecule, instead of two
molecules of trypsin, indicating that Arg76 and Arg87 but
not Lys44 are the two reactive sites of APIB. In order to further
confirm this result , more mutants of APIB (K44P-R76P-APIB,
K44P-R87P-APIB, R76P-R87P-APIB)
were designed, in each mutant only one of the three possible reactive sites
remained unchanged. Both the mutants K44P-R76P-APIB and K44P-R87P-APIB
could only inhibit one molecule of trypsin, while R76P-R87P-APIB
could no longer inhibit trypsin intensively, thus the residues Arg76
and Arg87 are definitely the reactive sites of APIB. Their K
were measured to be 0.39 nmol/L and 0.47 nmol/L, respectively. The mutant R87L-APIB
lost about half activity towards trypsin but could inhibit one molecule of
chymotrypsin as the wide type APIA did, indicating that Leu87 was
the reactive site towards trypsin but could inhibit one molecule of APIA for
inhibiting chymotrypsin.
Key words arrowhead proteinase inhibitor； site directed mutagenesis； inhibitory activity； reactive site
*Corresponding author： Tel, 86-21-64374430; Fax,
86-21-64338357; e-mail, [email protected]