Transient kinetics of ligand binding and role of the C-terminus in the dUTPase from equine infectious anemia virus

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Transient kinetics of ligand binding and role of the C-terminus in the dUTPase from equine infectious anemia virus. / Nord, Johan; Kiefer, Martin; Adolph, Hans-Werner; Zeppezauer, Michael M; Nyman, Per-Olof.

In: FEBS Letters, Vol. 472, No. 2-3, 2000, p. 312-316.

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Nord, Johan ; Kiefer, Martin ; Adolph, Hans-Werner ; Zeppezauer, Michael M ; Nyman, Per-Olof. / Transient kinetics of ligand binding and role of the C-terminus in the dUTPase from equine infectious anemia virus. In: FEBS Letters. 2000 ; Vol. 472, No. 2-3. pp. 312-316.

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TY - JOUR

T1 - Transient kinetics of ligand binding and role of the C-terminus in the dUTPase from equine infectious anemia virus

AU - Nord, Johan

AU - Kiefer, Martin

AU - Adolph, Hans-Werner

AU - Zeppezauer, Michael M

AU - Nyman, Per-Olof

PY - 2000

Y1 - 2000

N2 - Transient kinetics of the equine infectious anemia virus deoxyuridine 5'-triphosphate nucleotide hydrolase were characterized by monitoring the fluorescence of the protein. Rate constants for the association and dissociation of substrate and inhibitors were determined and found to be consistent with a one-step mechanism for substrate binding. A C-terminal part of the enzyme presumed to be flexible was removed by limited trypsinolysis. As a result, the activity of the dUTPase was completely quenched, but the rate constants and fluorescent signal of the truncated enzyme were affected only to a minor degree. We conclude that the flexible C-terminus is not a prerequisite for substrate binding, but indispensable for catalysis.

AB - Transient kinetics of the equine infectious anemia virus deoxyuridine 5'-triphosphate nucleotide hydrolase were characterized by monitoring the fluorescence of the protein. Rate constants for the association and dissociation of substrate and inhibitors were determined and found to be consistent with a one-step mechanism for substrate binding. A C-terminal part of the enzyme presumed to be flexible was removed by limited trypsinolysis. As a result, the activity of the dUTPase was completely quenched, but the rate constants and fluorescent signal of the truncated enzyme were affected only to a minor degree. We conclude that the flexible C-terminus is not a prerequisite for substrate binding, but indispensable for catalysis.

KW - C-terminus

KW - Deoxyuridine 5'-triphosphate nucleotide hydrolase

KW - Deoxyuridine

KW - Equine infectious anemia virus

KW - Ligand binding

KW - Pre-steady-state kinetics

KW - Rate constant

U2 - 10.1016/S0014-5793(00)01453-8

DO - 10.1016/S0014-5793(00)01453-8

M3 - Article

VL - 472

SP - 312

EP - 316

JO - FEBS Letters

T2 - FEBS Letters

JF - FEBS Letters

SN - 1873-3468

IS - 2-3

ER -