Slow step after bond-breaking by porcine pepsin identified using solvent deuterium isotope effects

Journal Article · Mon Apr 15 04:00:00 EDT 1991 · Biochemical and Biophysical Research Communications; (USA)

DOI:https://doi.org/10.1016/0006-291X(91)90890-J· OSTI ID:5583566

Rebholz, K L; Northrop, D B ^[1]

---

1. Univ. of Wisconsin, Madison (USA)

The relatively fast artificial substrate Leu-Ser-rho-nitro-Phe-Nle-Ala-Leu-OMe generates a solvent isotope effect of 1.51 +/- 0.02 only on the maximal velocity of peptide hydrolysis catalyzed by porcine pepsin. The absence of an isotope effect on V/K places the isotopically-sensitive step after peptide bond cleavage and the release of the first product. Reprotonation of the active site aspartic carboxyls is proposed as the most likely interpretation of this observation. Structural and kinetic similarities between pepsin and other aspartic proteinases, including the therapeutically important targets HIV protease and renin, suggest a similar slow reprotonation step after catalysis. This mechanistic feature has important implications regarding inhibitor design; if most of the enzymes are present in a product-release form during steady-state turnover, then perhaps inhibitors should be designed as product analogs instead of substrate analogs.

• OSTI ID: 5583566
• Journal Information: Biochemical and Biophysical Research Communications; (USA), Journal Name: Biochemical and Biophysical Research Communications; (USA) Vol. 176:1; ISSN BBRCA; ISSN 0006-291X
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
550501 -- Metabolism-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACID PROTEINASES
AMINO ACID SEQUENCE
AMINO ACIDS
ANIMALS
ASPARTIC ACID
CARBOXYLIC ACIDS
DEUTERIUM
DOMESTIC ANIMALS
ENZYME ACTIVITY
ENZYMES
HYDROGEN ISOTOPES
HYDROLASES
ISOTOPE APPLICATIONS
ISOTOPE EFFECTS
ISOTOPES
LIGHT NUCLEI
MAMMALS
MEMBRANE PROTEINS
METABOLISM
MOLECULAR STRUCTURE
NUCLEI
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
PEPSIN
PEPTIDE HYDROLASES
PEPTIDES
PROTEINS
RECEPTORS
STABLE ISOTOPES
STEADY-STATE CONDITIONS
SUBSTRATES
SWINE
TRACER TECHNIQUES
VERTEBRATES