Kinetic and structural effects of mutations of the catalytic amino- terminal proline in 4-oxalocrotonate tautomerase

Robert M. Czerwinski, William H. Johnson, Christian P. Whitman, Thomas K Harris, Chitrananda Abeygunawardana, Albert S. Mildvan

Research output: Contribution to journalArticle

46 Citations (Scopus)

Abstract

The catalytic general base, Pro-1, of the enzyme 4-oxalocrotonate tautomerase has been mutated to Gly, Ala, Va], and Leu, residues with aliphatic side chains. The Val mutant was partially (55%) processed by removal of the amino-terminal methionine to yield P1V/M1P2V, while the Leu mutant was not processed and completely retained methionine (M1P2L). The M1P2L mutant lost 2300-fold in k(cat) with no change in K(m), and the residual activity of the unresolvable P1V/M1P2V mixture could be explained by the summation of two activities, one equal to that of M1P2L and the other equal to that of the PIG mutant. The P1G and P1A mutants showed 76- and 58- fold decreases in k(cat) and much smaller decreases in K(m) of 4- and 2.8- fold, respectively. The dissociation constant of the substrate analog cis,cis-muconate decreased 1.7-fold in the P1G mutant as determined by NMR titration, 2D 1H-15N HSQC spectra and 3D 1H-15N NOESY HSQC spectra of the 15N-labeled P1G mutant showed no structural differences from the wild- type enzyme except for small changes in backbone 15N and NH chemical shifts at the active site. Both the P1G and P1A mutants showed no change in overall conformation by circular dichroic spectroscopy. Both mutants and the wild- type enzyme generate the S-enantiomer of the product [5-2H]-2-oxo-3- hexenedioate with comparable stereoselectivities indicating a largely intact active site. The P1G and P1A mutants showed 10- and 4-fold decreases, respectively, in catalysis of exchange of the C3 proton of the substrate 2- oxo-1,6-hexanedioate, consistent with the lower basicities of Gly-1 and Ala- 1 compared to Pro-1. The pH dependences of k(cat)/K(m) for the P1G and P1A mutants revealed pK(a) values of the general base of 5.3 and 5.9, respectively. NMR titration of the uniformly 15N-labeled P1G mutant showed the pK(a) of Gly-1 to be ≤5.6, in agreement with the kinetic data. As with the wild-type enzyme, the active site environments on the P1G and P1A mutants lower the pK(a) of the general base by at least 2.5 units. It is concluded that the 2 order of magnitude decreases in k(cat) in the P1G and P1A mutants result from both a decrease in basicity and an increase in flexibility of the general base. The greater 103.4-fold decrease in k(cat) found with the presence of an additional residue at the amino-terminus is ascribed to either the complete blockage or the drastically altered position of the general base.

Original languageEnglish
Pages (from-to)14551-14560
Number of pages10
JournalBiochemistry
Volume36
Issue number47
DOIs
StatePublished - Nov 25 1997
Externally publishedYes

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Proline
Catalytic Domain
Mutation
Kinetics
Enzymes
Alkalinity
Titration
Methionine
Nuclear magnetic resonance
Stereoselectivity
Enantiomers
Chemical shift
Substrates
Catalysis
Conformations
Protons
Spectrum Analysis
Ion exchange
Spectroscopy
4-oxalocrotonate tautomerase

ASJC Scopus subject areas

  • Biochemistry

Cite this

Czerwinski, R. M., Johnson, W. H., Whitman, C. P., Harris, T. K., Abeygunawardana, C., & Mildvan, A. S. (1997). Kinetic and structural effects of mutations of the catalytic amino- terminal proline in 4-oxalocrotonate tautomerase. Biochemistry, 36(47), 14551-14560. https://doi.org/10.1021/bi971545h

Kinetic and structural effects of mutations of the catalytic amino- terminal proline in 4-oxalocrotonate tautomerase. / Czerwinski, Robert M.; Johnson, William H.; Whitman, Christian P.; Harris, Thomas K; Abeygunawardana, Chitrananda; Mildvan, Albert S.

In: Biochemistry, Vol. 36, No. 47, 25.11.1997, p. 14551-14560.

Research output: Contribution to journalArticle

Czerwinski, RM, Johnson, WH, Whitman, CP, Harris, TK, Abeygunawardana, C & Mildvan, AS 1997, 'Kinetic and structural effects of mutations of the catalytic amino- terminal proline in 4-oxalocrotonate tautomerase', Biochemistry, vol. 36, no. 47, pp. 14551-14560. https://doi.org/10.1021/bi971545h
Czerwinski, Robert M. ; Johnson, William H. ; Whitman, Christian P. ; Harris, Thomas K ; Abeygunawardana, Chitrananda ; Mildvan, Albert S. / Kinetic and structural effects of mutations of the catalytic amino- terminal proline in 4-oxalocrotonate tautomerase. In: Biochemistry. 1997 ; Vol. 36, No. 47. pp. 14551-14560.
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T1 - Kinetic and structural effects of mutations of the catalytic amino- terminal proline in 4-oxalocrotonate tautomerase

AU - Czerwinski, Robert M.

AU - Johnson, William H.

AU - Whitman, Christian P.

AU - Harris, Thomas K

AU - Abeygunawardana, Chitrananda

AU - Mildvan, Albert S.

PY - 1997/11/25

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N2 - The catalytic general base, Pro-1, of the enzyme 4-oxalocrotonate tautomerase has been mutated to Gly, Ala, Va], and Leu, residues with aliphatic side chains. The Val mutant was partially (55%) processed by removal of the amino-terminal methionine to yield P1V/M1P2V, while the Leu mutant was not processed and completely retained methionine (M1P2L). The M1P2L mutant lost 2300-fold in k(cat) with no change in K(m), and the residual activity of the unresolvable P1V/M1P2V mixture could be explained by the summation of two activities, one equal to that of M1P2L and the other equal to that of the PIG mutant. The P1G and P1A mutants showed 76- and 58- fold decreases in k(cat) and much smaller decreases in K(m) of 4- and 2.8- fold, respectively. The dissociation constant of the substrate analog cis,cis-muconate decreased 1.7-fold in the P1G mutant as determined by NMR titration, 2D 1H-15N HSQC spectra and 3D 1H-15N NOESY HSQC spectra of the 15N-labeled P1G mutant showed no structural differences from the wild- type enzyme except for small changes in backbone 15N and NH chemical shifts at the active site. Both the P1G and P1A mutants showed no change in overall conformation by circular dichroic spectroscopy. Both mutants and the wild- type enzyme generate the S-enantiomer of the product [5-2H]-2-oxo-3- hexenedioate with comparable stereoselectivities indicating a largely intact active site. The P1G and P1A mutants showed 10- and 4-fold decreases, respectively, in catalysis of exchange of the C3 proton of the substrate 2- oxo-1,6-hexanedioate, consistent with the lower basicities of Gly-1 and Ala- 1 compared to Pro-1. The pH dependences of k(cat)/K(m) for the P1G and P1A mutants revealed pK(a) values of the general base of 5.3 and 5.9, respectively. NMR titration of the uniformly 15N-labeled P1G mutant showed the pK(a) of Gly-1 to be ≤5.6, in agreement with the kinetic data. As with the wild-type enzyme, the active site environments on the P1G and P1A mutants lower the pK(a) of the general base by at least 2.5 units. It is concluded that the 2 order of magnitude decreases in k(cat) in the P1G and P1A mutants result from both a decrease in basicity and an increase in flexibility of the general base. The greater 103.4-fold decrease in k(cat) found with the presence of an additional residue at the amino-terminus is ascribed to either the complete blockage or the drastically altered position of the general base.

AB - The catalytic general base, Pro-1, of the enzyme 4-oxalocrotonate tautomerase has been mutated to Gly, Ala, Va], and Leu, residues with aliphatic side chains. The Val mutant was partially (55%) processed by removal of the amino-terminal methionine to yield P1V/M1P2V, while the Leu mutant was not processed and completely retained methionine (M1P2L). The M1P2L mutant lost 2300-fold in k(cat) with no change in K(m), and the residual activity of the unresolvable P1V/M1P2V mixture could be explained by the summation of two activities, one equal to that of M1P2L and the other equal to that of the PIG mutant. The P1G and P1A mutants showed 76- and 58- fold decreases in k(cat) and much smaller decreases in K(m) of 4- and 2.8- fold, respectively. The dissociation constant of the substrate analog cis,cis-muconate decreased 1.7-fold in the P1G mutant as determined by NMR titration, 2D 1H-15N HSQC spectra and 3D 1H-15N NOESY HSQC spectra of the 15N-labeled P1G mutant showed no structural differences from the wild- type enzyme except for small changes in backbone 15N and NH chemical shifts at the active site. Both the P1G and P1A mutants showed no change in overall conformation by circular dichroic spectroscopy. Both mutants and the wild- type enzyme generate the S-enantiomer of the product [5-2H]-2-oxo-3- hexenedioate with comparable stereoselectivities indicating a largely intact active site. The P1G and P1A mutants showed 10- and 4-fold decreases, respectively, in catalysis of exchange of the C3 proton of the substrate 2- oxo-1,6-hexanedioate, consistent with the lower basicities of Gly-1 and Ala- 1 compared to Pro-1. The pH dependences of k(cat)/K(m) for the P1G and P1A mutants revealed pK(a) values of the general base of 5.3 and 5.9, respectively. NMR titration of the uniformly 15N-labeled P1G mutant showed the pK(a) of Gly-1 to be ≤5.6, in agreement with the kinetic data. As with the wild-type enzyme, the active site environments on the P1G and P1A mutants lower the pK(a) of the general base by at least 2.5 units. It is concluded that the 2 order of magnitude decreases in k(cat) in the P1G and P1A mutants result from both a decrease in basicity and an increase in flexibility of the general base. The greater 103.4-fold decrease in k(cat) found with the presence of an additional residue at the amino-terminus is ascribed to either the complete blockage or the drastically altered position of the general base.

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