Mechanistic study of protein phosphatase-1 (PP1), a catalytically promiscuous enzyme.
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.
McWhirter
Claire
C
Centre for Chemical Biology, Department of Chemistry, University of Sheffield, Sheffield, UK S3 7HF.
Lund
Elizabeth A
EA
Tanifum
Eric A
EA
Feng
Guoqiang
G
Sheikh
Qaiser I
QI
Hengge
Alvan C
AC
Williams
Nicholas H
NH
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
D009596:Nitrophenols; D009943:Organophosphorus Compounds; C008644:nitrophenylphosphate; D054645:Protein Phosphatase 1
Biochemistry. 1996 Jun 4;35(22):7084-92
8679534
Biochemistry. 2001 May 15;40(19):5691-9
11341834
Biochemistry. 2004 Aug 17;43(32):10547-59
15301552
J Mol Biol. 1995 Dec 15;254(5):942-59
7500362
Biochemistry. 1996 May 21;35(20):6276-82
8639569
Nature. 1995 Aug 31;376(6543):745-53
7651533
J Am Chem Soc. 2002 Sep 18;124(37):10964-5
12224928
Proc Natl Acad Sci U S A. 2003 May 13;100(10):5607-10
12721374
Biochemistry. 1997 Jun 24;36(25):7928-36
9201938
Biochemistry. 1995 Dec 12;34(49):16088-96
8519766
Biochemistry. 1995 Oct 31;34(43):13982-7
7577995
J Am Chem Soc. 2007 May 2;129(17):5760-5
17411045
Nature. 1995 Dec 7;378(6557):641-4
8524402
Eur J Biochem. 1996 Jul 15;239(2):317-25
8706735
Biochemistry. 1999 Mar 16;38(11):3386-92
10079083
Biochemistry. 1991 Apr 16;30(15):3634-9
2015221
J Biol Chem. 2004 Aug 6;279(32):33992-9
15155720
Science. 1995 Jun 9;268(5216):1489-92
7770774
Biochemistry. 2002 Mar 5;41(9):3207-25
11863460
J Biol Chem. 1994 Mar 18;269(11):8140-5
8132539
Acc Chem Res. 2002 Feb;35(2):105-12
11851388
Biochem J. 1991 Apr 1;275 ( Pt 1):233-9
1850239
J Am Chem Soc. 2007 Aug 8;129(31):9789-98
17630738
J Am Chem Soc. 2006 Feb 1;128(4):1293-303
16433548
Spectrochim Acta A Mol Biomol Spectrosc. 2007 Feb;66(2):413-8
16843706
Biochemistry. 1997 Aug 19;36(33):10185-91
9254616
J Biol Inorg Chem. 2004 Dec;9(8):973-86
15452776
Proc Natl Acad Sci U S A. 1997 Apr 15;94(8):3530-5
9108010