Substrate and inhibitor studies with human gastric aspartic proteinases.
The separation of pepsin isoenzymes 1, 2, 3 and 5 (gastricsin) in human gastric juice was effected by chromatography on Mono Q ion-exchanger, and slow-moving proteinase was purified to homogeneity by using a modified procedure incorporating a novel affinity-chromatography step. The pH-activity profiles of these enzymes with mucus glycoprotein and basement-membrane substrates were determined; the profiles for pepsin 2 were noticeably different, and, in general, the pH optima for the hydrolysis of basement membrane were more acidic. Pepsin 1 expressed larger specificity constants (kcat./Km) than pepsin 3 with a series of synthetic peptide substrates, reflecting greater binding (smaller Km) by pepsin 1. Inhibitor studies at pH 1.7 and 4.5 with a series of P2-substituted lactoyl-pepstatins implied that valine at position P2 was optimal for inhibiting pepsins 1, 2 and 3 but detrimental for pepsin 5, whereas lysine at position P2 was tolerated well by pepsin 5 but not by pepsins 1, 2 and 3. The potency of lactoyl-pepstatin with lysine at position P2 did not increase as a function of pH. P2-substituted lactoyl-pepstatins failed to show any inhibitory selectivity among pepsins 1, 2 and 3.
Baxter
A
A
Department of Biochemistry, Glaxo Group Research Ltd., Greenford, Middx., U.K.
Campbell
C J
CJ
Grinham
C J
CJ
Keane
R M
RM
Lawton
B C
BC
Pendlebury
J E
JE
D001485:Basement Membrane / Q000378:metabolism; D005753:Gastric Mucosa / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D009077:Mucins / Q000378:metabolism; D010434:Pepsin A / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D013329:Structure-Activity Relationship
D007527:Isoenzymes; D009077:Mucins; D010434:Pepsin A; C020557:gastricsin