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Bovine liver dihydropyrimidine amidohydrolase: pH dependencies of the steady-state kinetic and proton relaxation rate properties of the Mn(II)-containing enzyme.

Abstract：

:The essential Zn(II) in bovine liver dihydropyrimidine amidohydrolase (DHPase) was removed by incubation with 2,6-dipicolinic acid and replaced with Mn(II). Electron paramagnetic resonance studies of Mn(II) binding show that there are four binding sites per tetramer, and the dissociation constant at pH 7.5 is 13.5 microM. The substitution of Mn(II) for Zn(II) increases the specific activity of the enzyme approximately sixfold but has only a small effect (twofold increase) on the Km for 5-bromo-5,6-dihydrouracil (BrH2Ura). The pH dependence of the catalytic properties of Mn(II)-DHPase is the same as for the Zn(II) enzyme (Lee, M., Cowling, R., Sander, E., and Pettigrew, D. (1986) Arch. Biochem. Biophys. 248, 368-378). The pH dependence is well described in terms of the ionization of a single group with a pK of about 6 in the free enzyme. The ionization of this group is required for catalytic activity. The substitution of Mn(II) for Zn(II) does not affect the pH dependence of DHPase catalysis and therefore strongly suggests that the ionizable group is an amino acid residue at or near the active site, rather than a metal-bound water molecule. The pH dependence of the enhancement of the paramagnetic effect of the DHPase-Mn complex on the relaxation rate of the solvent water protons also is well described in terms of the ionization of a single group with a pK of about 6. Ionization of the group which is involved in catalysis also perturbs the environment of the bound Mn(II). The ionization of the active site group does not affect the number of exchangeable water molecules but does affect the symmetry of the environment of the bound Mn(II) and its electron relaxation.

journal_name

Arch Biochem Biophys

journal_title

Archives of biochemistry and biophysics

authors

Lee MH,Pettigrew DW,Sander EG,Nowak T

doi

10.1016/0003-9861(87)90526-1

subject

Has Abstract

pub_date

1987-12-01 00:00:00

pages

597-604

issue

eissn

0003-9861

issn

1096-0384

pii

0003-9861(87)90526-1

journal_volume

259

pub_type

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Bovine liver dihydropyrimidine amidohydrolase: pH dependencies of the steady-state kinetic and proton relaxation rate properties of the Mn(II)-containing enzyme.