title: Mechanistic study of protein phosphatase-1 (PP1), a catalytically promiscuous enzyme.
abstract: The reaction catalyzed by the protein phosphatase-1 (PP1) has been examined by linear free energy relationships and kinetic isotope effects. With the substrate 4-nitrophenyl phosphate (4NPP), the reaction exhibits a bell-shaped pH-rate profile for kcat/KM indicative of catalysis by both acidic and basic residues, with kinetic pKa values of 6.0 and 7.2. The enzymatic hydrolysis of a series of aryl monoester substrates yields a Brønsted beta(lg) of -0.32, considerably less negative than that of the uncatalyzed hydrolysis of monoester dianions (-1.23). Kinetic isotope effects in the leaving group with the substrate 4NPP are (18)(V/K) bridge = 1.0170 and (15)(V/K) = 1.0010, which, compared against other enzymatic KIEs with and without general acid catalysis, are consistent with a loose transition state with partial neutralization of the leaving group. PP1 also efficiently catalyzes the hydrolysis of 4-nitrophenyl methylphosphonate (4NPMP). The enzymatic hydrolysis of a series of aryl methylphosphonate substrates yields a Brønsted beta(lg) of -0.30, smaller than the alkaline hydrolysis (-0.69) and similar to the beta(lg) measured for monoester substrates, indicative of similar transition states. The KIEs and the beta(lg) data point to a transition state for the alkaline hydrolysis of 4NPMP that is similar to that of diesters with the same leaving group. For the enzymatic reaction of 4NPMP, the KIEs are indicative of a transition state that is somewhat looser than the alkaline hydrolysis reaction and similar to the PP1-catalyzed monoester reaction. The data cumulatively point to enzymatic transition states for aryl phosphate monoester and aryl methylphosphonate hydrolysis reactions that are much more similar to one another than the nonenzymatic hydrolysis reactions of the two substrates.
authors.affiliation: Centre for Chemical Biology, Department of Chemistry, University of Sheffield, Sheffield, UK S3 7HF.
authors.forename: Claire
authors.initials: C
authors.lastname: McWhirter
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Lund
authors.forename: Eric A
authors.initials: EA
authors.lastname: Tanifum
authors.forename: Guoqiang
authors.initials: G
authors.lastname: Feng
authors.forename: Qaiser I
authors.initials: QI
authors.lastname: Sheikh
authors.forename: Alvan C
authors.initials: AC
authors.lastname: Hengge
authors.forename: Nicholas H
authors.initials: NH
authors.lastname: Williams
mesh_terms: D002384:Catalysis; D015394:Molecular Structure; D009596:Nitrophenols / Q000737:chemistry / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism; D054645:Protein Phosphatase 1 / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D009596:Nitrophenols; D009943:Organophosphorus Compounds; C008644:nitrophenylphosphate; D054645:Protein Phosphatase 1
references.citation: Biochemistry. 1996 Jun 4;35(22):7084-92
references.pmid: 8679534
references.citation: Biochemistry. 2001 May 15;40(19):5691-9
references.pmid: 11341834
references.citation: Biochemistry. 2004 Aug 17;43(32):10547-59
references.pmid: 15301552
references.citation: J Mol Biol. 1995 Dec 15;254(5):942-59
references.pmid: 7500362
references.citation: Biochemistry. 1996 May 21;35(20):6276-82
references.pmid: 8639569
references.citation: Nature. 1995 Aug 31;376(6543):745-53
references.pmid: 7651533
references.citation: J Am Chem Soc. 2002 Sep 18;124(37):10964-5
references.pmid: 12224928
references.citation: Proc Natl Acad Sci U S A. 2003 May 13;100(10):5607-10
references.pmid: 12721374
references.citation: Biochemistry. 1997 Jun 24;36(25):7928-36
references.pmid: 9201938
references.citation: Biochemistry. 1995 Dec 12;34(49):16088-96
references.pmid: 8519766
references.citation: Biochemistry. 1995 Oct 31;34(43):13982-7
references.pmid: 7577995
references.citation: J Am Chem Soc. 2007 May 2;129(17):5760-5
references.pmid: 17411045
references.citation: Nature. 1995 Dec 7;378(6557):641-4
references.pmid: 8524402
references.citation: Eur J Biochem. 1996 Jul 15;239(2):317-25
references.pmid: 8706735
references.citation: Biochemistry. 1999 Mar 16;38(11):3386-92
references.pmid: 10079083
references.citation: Biochemistry. 1991 Apr 16;30(15):3634-9
references.pmid: 2015221
references.citation: J Biol Chem. 2004 Aug 6;279(32):33992-9
references.pmid: 15155720
references.citation: Science. 1995 Jun 9;268(5216):1489-92
references.pmid: 7770774
references.citation: Biochemistry. 2002 Mar 5;41(9):3207-25
references.pmid: 11863460
references.citation: J Biol Chem. 1994 Mar 18;269(11):8140-5
references.pmid: 8132539
references.citation: Acc Chem Res. 2002 Feb;35(2):105-12
references.pmid: 11851388
references.citation: Biochem J. 1991 Apr 1;275 ( Pt 1):233-9
references.pmid: 1850239
references.citation: J Am Chem Soc. 2007 Aug 8;129(31):9789-98
references.pmid: 17630738
references.citation: J Am Chem Soc. 2006 Feb 1;128(4):1293-303
references.pmid: 16433548
references.citation: Spectrochim Acta A Mol Biomol Spectrosc. 2007 Feb;66(2):413-8
references.pmid: 16843706
references.citation: Biochemistry. 1997 Aug 19;36(33):10185-91
references.pmid: 9254616
references.citation: J Biol Inorg Chem. 2004 Dec;9(8):973-86
references.pmid: 15452776
references.citation: Proc Natl Acad Sci U S A. 1997 Apr 15;94(8):3530-5
references.pmid: 9108010