/sup 18/O isotope effect in /sup 13/C nuclear magnetic resonance spectroscopy. Part 9. Hydrolysis of benzyl phosphate by phosphatase enzymes and in acidic aqueous solutions

Journal Article · Tue Dec 25 23:00:00 EST 1984 · J. Am. Chem. Soc.; (United States)

DOI:https://doi.org/10.1021/ja00338a026· OSTI ID:5861463

Parente, J E; Risley, J M; Van Etten, R L

The /sup 18/O isotope-induced shifts in /sup 13/C and /sup 31/P nuclear magnetic resonance (NMR) spectroscopy were used to establish the position of bond cleavage in the phosphatase-catalyzed and acid-catalyzed hydrolysis reactions of benzyl phosphate. The application of the /sup 18/O-isotope effect in NMR spectroscopy affords a continuous, nondestructive assay method for following the kinetics and position of bond cleavage in the hydrolytic process. The technique provides advantages over most discontinuous methods in which the reaction components must be isolated and converted to volatile derivatives prior to analysis. In the present study, (..cap alpha..-/sup 13/C,ester-/sup 18/O)benzyl phosphate and (ester-/sup 18/O)benzyl phosphate were synthesized for use in enzymatic and nonenzymatic studies. Hydrolysis reactions catalyzed by the alkaline phosphatase from E. coli and by the acid phosphatases isolated from human prostate and human liver were all accompanied by cleavage of the substrate phosphorus-oxygen bond consistent with previously postulated mechanisms involving covalent phosphoenzyme intermediates. An extensive study of the acid-catalyzed hydrolysis of benzyl phosphate at 75/sup 0/C revealed that the site of bond cleavage is dependent on pH. At pH less than or equal to 1.3, the hydrolysis proceeds with C-O bond cleavage; at 1.3 < pH < 2.0, there is a mixture of C-O and P-O bond scission, the latter progressively predominating as the pH is raised; at pH greater than or equal to 2.0, the hydrolysis proceeds with exclusive P-O bond scission. (S)-(+)-(..cap alpha..-/sup 2/H)Benzyl phosphate was also synthesized. Hydrolysis of this chiral benzyl derivative demonstrated that the acid-catalyzed C-O bond scission of benzyl phosphate proceeds by an A-1 (S/sub N/1) mechanism with 70% racemization and 30% inversion at carbon. 37 references, 4 figures, 2 tables.

• Research Organization: Purdue Univ., West Lafayette, IN
• OSTI ID: 5861463
• Journal Information: J. Am. Chem. Soc.; (United States), Journal Name: J. Am. Chem. Soc.; (United States) Vol. 106:26; ISSN JACSA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACID HYDROLYSIS
ACID PHOSPHATASE
ALKALINE PHOSPHATASE
ANIMAL CELLS
AQUEOUS SOLUTIONS
AROMATICS
BACTERIA
BODY
CHEMICAL ANALYSIS
CHEMICAL REACTION KINETICS
CHEMICAL REACTIONS
DATA
DECOMPOSITION
DISPERSIONS
ENZYMATIC HYDROLYSIS
ENZYME ACTIVITY
ENZYMES
ESCHERICHIA COLI
ESTERASES
ESTERS
EXPERIMENTAL DATA
GLANDS
HYDROLASES
HYDROLYSIS
INCUBATION
INFORMATION
ISOTOPE APPLICATIONS
KINETICS
LIVER CELLS
LYSIS
MALE GENITALS
MICROORGANISMS
MIXTURES
MONITORING
NMR SPECTRA
NUMERICAL DATA
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
ORGANS
PH VALUE
PHOSPHATASES
PHOSPHORIC ACID ESTERS
PROSTATE
QUALITATIVE CHEMICAL ANALYSIS
REACTION KINETICS
SOLUTIONS
SOLVOLYSIS
SOMATIC CELLS
SPECTRA
SPECTRAL SHIFT
TRACER TECHNIQUES