Catalysis by human leukocyte elastase: proton inventory as a mechanistic probe

Journal Article · Tue Mar 10 04:00:00 EST 1987 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00379a016· OSTI ID:6478383

Stein, R L; Strimpler, A M; Hori, H; Powers, J C

Proton inventories (rate measurements in mixtures of H/sub 2/O and D/sub 2/O) were determined for the human leukocyte elastase catalyzed hydrolyses of thiobenzyl esters and p-nitroanilides of the peptides MeOSuc-Val, MeOSuc-Alan-Pro-Val (n = 0-2), and MeOSuc-Alan-Pro-Ala (n = 1 or 2). The dependencies of k2/Ks on mole fraction of solvent deuterium for the p-nitroanilides are dome-shaped and were fit to a model that incorporates the mechanistic features of generalized solvent reorganization when substrate binds to enzyme and partial rate limitation of k2/Ks by physical and chemical steps. The proton inventories for the deacylation of MeOSuc-Val-HLE and MeOSuc-Pro-Val-HLE are linear while those for the deacylation of MeOSuc-Ala-Pro-Val-HLE and MeOSuc-Ala-Ala-Pro-Val-HLE are bowl-shaped and could be fit to a quadratic dependence of rate on mole fraction of deuterium. These results are interpreted to suggest that the correct operation of the catalytic triad is dependent on substrate structure. Minimal substrates, which cannot interact with elastase at remote subsites, are hydrolyzed via a mechanism involving simple general-base catalysis by the active site histidine and transfer of a single proton in the rate-limiting transition state. In contrast, tri- and tetrapeptide substrates, which are able to interact at remote subsites, are hydrolyzed by a more complex mechanism of protolytic catalysis involving full functioning of the catalytic triad and transfer of two protons in the rate-limiting transition state. Finally, the proton inventories for the deacylation of MeOSuc-Ala-Pro-Ala-HLE and MeOSuc-Ala-Ala-Pro-Ala-HLE are dome-shaped and suggest that the chemical events of acyl-enzyme hydrolysis are only partially rate limiting for these reactions and that some other physical step is also partially rate limiting.

• Research Organization: Stuart Pharmaceuticals, Wilmington, DE
• OSTI ID: 6478383
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 5; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
BARYONS
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY FLUIDS
CATALYSIS
CHEMICAL REACTIONS
DECOMPOSITION
DEUTERIUM
ELEMENTARY PARTICLES
FERMIONS
HADRONS
HEAVY WATER
HYDROGEN COMPOUNDS
HYDROGEN ISOTOPES
HYDROLYSIS
ISOTOPES
KINETICS
LEUKOCYTES
LIGHT NUCLEI
LYSIS
MATERIALS
MEASURING METHODS
NITRO COMPOUNDS
NUCLEI
NUCLEONS
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OXYGEN COMPOUNDS
PROTONS
REACTION KINETICS
SOLVOLYSIS
STABLE ISOTOPES
THERMODYNAMICS
WATER