Ala217 is Important for the Catalytic Function and Autoactivation of Prostate‐Specific Human Kallikrein 2

• Published in: European journal of biochemistry Vol. 246; no. 2; pp. 440 - 446
• Main Authors: Mikolajczyk, Stephen D., Millar, Lisa S., Marker, Kathy M., Grauer, Lana S., Goel, Amita, Cass, Michelle M. J., Kumar, Abhay, Saedi, Mohammad S.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01.06.1997
• Subjects: Alanine - metabolism; Amino Acid Sequence; Animals; Base Sequence; Catalysis; Cell Line; Cloning, Molecular; Cricetinae; DNA, Complementary; Enzyme Activation; human glandular kallikrein; Humans; Kallikreins - genetics; Kallikreins - isolation & purification; Kallikreins - metabolism; Kinetics; Male; Molecular Sequence Data; Mutagenesis; Prostate - enzymology; prostate cancer; prostate‐specific antigen; serine protease; Tissue Kallikreins
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1997.00440.x

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 Km of 4200 μM compared to 11 μM for wild‐type hK2. The kM 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.