Subsite specificities of granzyme M: a study of inhibitors and newly synthesized thiobenzyl ester substrates.
Granzyme M is a member of a family of granule serine proteases that participate in target cell death initiated by cytotoxic lymphocytes. The enzyme is almost exclusively expressed in NK cell types. Granzyme M cleaves at the carboxy side of amino acids with long, hydrophobic side chains like Met, Leu, and Nle. To further study the substrate specificity of the enzyme, a series of peptide thiobenzyl esters was synthesized. The hydrolysis of the substrates with murine and human recombinant forms of granzyme M was observed. The results show that the enzyme has a strong preference for Pro at the P2 position and Ala, Ser, or Asp at the P3 position. These results suggest that the protein residues of the S2 and S3 subsites form important binding interactions that aid in the selection of specific natural substrates for granzyme M. A series of inhibitors was also tested with granzyme M. None of the inhibitors were effective inactivators of granzyme M, including the general serine protease inhibitor, 3,4-dichloroisocoumarin, which is usually a potent inactivator of serine proteases. This suggests that inhibition of granzyme M may be difficult. Also reported for the first time is the method utilized to isolate granzyme M used in this and previous publications. The observations in this paper will be valuable in development of new potent inhibitors for granzyme M as well as assist in determining the biological function of the enzyme.
Rukamp
Brian J
BJ
The School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332-0400, USA.
Kam
Chih Min
CM
Natarajan
Sudah
S
Bolton
Brad W
BW
Smyth
Mark J
MJ
Kelly
Janice M
JM
Powers
James C
JC
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002460:Cell Line; D002918:Chymotrypsin / Q000378:metabolism; D053804:Granzymes; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D051379:Mice; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D012697:Serine Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D018411:Spodoptera; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology
D010455:Peptides; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D013438:Sulfhydryl Compounds; C509218:GZMM protein, human; D053804:Granzymes; C509219:Gzmm protein, mouse; C509220:Gzmm protein, rat; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C117608:alpha-chymotrypsin