Ala217 is important for the catalytic function and autoactivation of prostate-specific human kallikrein 2.
Prostate-specific human kallikrein, hK2, is a serine protease found in prostate tissues that has 78% amino acid sequence identity with prostate-specific antigen (PSA). We have previously reported the affinity purification of hK2 heterologously expressed in a hamster cell line and demonstrated an arginine-restricted substrate specificity. Here, we describe the cloning, expression, purification, and enzymatic activity of a mutant form of hK2 containing an alanine to valine substitution at residue 217 ([Val217]hK2). This mutant form was secreted into the serum-free spent media of recombinant cells as the stable proenzyme form ([Val217]phK2). Mild trypsin treatment was used to convert [Val217]phK2 to the active form, which had reduced catalytic function compared to the wild-type hK2. Kinetic studies using the chromogenic substrate D-H-Pro-Phe-Arg-4-nitroanilide showed that [Val217]hK2 has significantly decreased substrate binding, with a K(m) of 4200 microM compared to 11 microM for wild-type hK2. The k(cat) for [Val217]hK2 was more than 100-fold lower than for hK2. hK2, but not [Val217]hK2, was able to activate [Val217]phK2. [Val217]hK2 also showed altered specificity on a synthetic peptide substrate compared to wild-type hK2, which exhibited partial hydrolysis at a PSA chymotrypsin-like cleavage site as well as the trypsin-like site cleaved by hK2. These results indicate that Ala217 is a key residue affecting the catalytic properties of hK2.
Mikolajczyk
S D
SD
Hybritech Incorporated, San Diego, California 92196-9006, USA. sdmikolajczyk@beckman.com
Millar
L S
LS
Marker
K M
KM
Grauer
L S
LS
Goel
A
A
Cass
M M
MM
Kumar
A
A
Saedi
M S
MS
D000409:Alanine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary; D004789:Enzyme Activation; D006801:Humans; D007610:Kallikreins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011467:Prostate / Q000201:enzymology*; D020840:Tissue Kallikreins
D018076:DNA, Complementary; D007610:Kallikreins; D020840:Tissue Kallikreins; D000409:Alanine