Structure-activity relationships in the hydrolysis of substrates by the phosphotriesterase from Pseudomonas diminuta

Journal Article · Tue May 30 00:00:00 EDT 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00437a021· OSTI ID:5472690

Donarski, W J; Dumas, D P; Heitmeyer, D P; Lewis, V E; Raushel, F M ^[1]

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1. Texas A M Univ., College Station (USA)

The mechanism and substrate specificity of the phosphotriesterase from Pseudomonas diminuta have been examined. The enzyme hydrolyzes a large number of phosphotriester substrates in addition to paraoxon (diethyl p-nitrophenyl phosphate) and its thiophosphate analogue, parathion. The two ethyl groups in paraoxon can be changed to propyl and butyl groups, but the maximal velocity and Km values decrease substantially. The enzyme will not hydrolyze phosphomonoesters or -diesters. There is a linear correlation between enzymatic activity and the pKa of the phenolic leaving group for 16 paraoxon analogues. The beta value in the corresponding Bronsted plot is -0.8. No effect on either Vmax or Vmax/Km is observed when sucrose is used to increase the relative solvent viscosity by 3-fold. These results are consistent with rate-limiting phosphorus-oxygen bond cleavage. A plot of log V versus pH for the hydrolysis of paraoxon shows one enzymatic group that must be unprotonated for activity with a pKa of 6.1. The deuterium isotope effect by D2O on Vmax and Vmax/Km is 2.4 and 1.2, respectively, and the proton inventory is linear, which indicates that only one proton is in flight during the transition state. The inhibition patterns by the products are consistent with a random kinetic mechanism.

• OSTI ID: 5472690
• Journal Information: Biochemistry; (USA), Vol. 28:11; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
PHOSPHATASES
BIOCHEMICAL REACTION KINETICS
SUBSTRATES
SPECIFICITY
DEUTERIUM
HYDROLYSIS
ISOTOPE EFFECTS
PHENOLS
PROTONS
PSEUDOMONAS
STRUCTURE-ACTIVITY RELATIONSHIPS
AROMATICS
BACTERIA
BARYONS
CHEMICAL REACTIONS
DECOMPOSITION
ELEMENTARY PARTICLES
ENZYMES
ESTERASES
FERMIONS
HADRONS
HYDROGEN ISOTOPES
HYDROLASES
HYDROXY COMPOUNDS
ISOTOPES
KINETICS
LIGHT NUCLEI
LYSIS
MICROORGANISMS
NUCLEI
NUCLEONS
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
REACTION KINETICS
SOLVOLYSIS
STABLE ISOTOPES
550201* - Biochemistry- Tracer Techniques