Source of catalysis in the lactate dehydrogenase system. Ground-state interactions in the enzyme·substrate complex

Hua Deng, J. Zheng, A. Clarke, J. J. Holbrook, Robert Callender, J. W. Burgner

Research output: Contribution to journalArticle

66 Citations (Scopus)

Abstract

The Raman spectra of both the NAD-pyruvate and the pyridine aldehyde adenine dinucleotide (PAAD)-pyruvate bound to pig heart, pig muscle, and Bacillus stearothermophilus lactate dehydrogenases were measured and are nearly the same, which is consistent with the conserved shell of residues surrounding the active-site cavity in these enzymes. The symmetrical stretching mode of the pyruvate carboxylate group, found at 1398 cm-1, is shifted only slightly when complexed to these enzymes, which shows that the group remains ionized in the ion pair complex with Arg-171 on the enzyme. The vibrational mode for the carbonyl stretch of the bound pyruvate moiety is shifted about 35 cm-1 to a lower frequency than observed for the carbonyl of unliganded pyruvate in the bacterial enzyme because of polarization of the carbonyl bond. Thus, the bacterial enzyme shows the same substrate activation because of the C+-O- charge separation that was seen previously with the mammalian enzymes. On the basis of an empirical Badger-Bauer relationship between frequency shift and interaction enthalpy, this shift in frequency is equivalent to an approximately -14 to -17 kcal/mol interaction between the enzyme and the adduct C=O coordinate, a substantial part of which is an electrostatic interaction (hydrogen bond) between the C=O and the protonated His-195. Thus, while the C=O bond is polarized on the enzyme (which requires energy), the overall ground-state enthalpy of the carbonyl imidazolium part of the reaction coordinate is stabilized substantially relative to its value in solution, and this is the dominant enthalpic effect on the entire reaction coordinate since the other internal coordinates for the hydride transfer are not much affected during formation of the ternary complex. The total enthalpy of binding for pyruvate analogs to lactate dehydrogenase is nearly the same as the sum of local enthalpies for interactions between pyruvate's C=O and the protein. Thus, even though ligand binding may cause any number of protein conformational changes, the net binding enthalpy of these changes must reflect the sum of a number of large, mostly compensating effects. The Raman spectra of the PAAD-pyruvate adduct bound to two different sets of mutant forms of the bacterial enzyme also were measured. Mutation of Arg-109, which normally hydrogen bonds to the pyruvate C=O, to Gln-109, reduces the extent of C+-O- charge separation by about 4 kcal/mol. Similarly, mutation of Asp- 168, which normally forms an ion pair with His-195 in the presence of a carbonyl-containing substrate to either Asn-168 or Ala-168 also reduces charge separation but to a somewhat greater degree, 4 and 10 kcal/mol, respectively. The ground state for the carbonyl imidazolium interactions of the mutant complexes is thus destabilized relative to the wild-type enzyme, and yet, the height of the transition-state barrier must increase, which clearly indicates the height of the barrier must increase faster than the ground state is destabilized. This view is analyzed from a plot of the log of the hydride transfer step versus the change in frequency of the C=O stretch (or ground-state interaction enthalpy).

Original languageEnglish (US)
Pages (from-to)2297-2305
Number of pages9
JournalBiochemistry
Volume33
Issue number8
StatePublished - 1994
Externally publishedYes

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Catalysis
L-Lactate Dehydrogenase
Ground state
Pyruvic Acid
Enzymes
Enthalpy
Adenine
Aldehydes
Hydrides
Raman scattering
Hydrogen
Hydrogen bonds
Lactate Dehydrogenases
Swine
Ions
Mustelidae
Geobacillus stearothermophilus
Mutation
Bacilli
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Source of catalysis in the lactate dehydrogenase system. Ground-state interactions in the enzyme·substrate complex. / Deng, Hua; Zheng, J.; Clarke, A.; Holbrook, J. J.; Callender, Robert; Burgner, J. W.

In: Biochemistry, Vol. 33, No. 8, 1994, p. 2297-2305.

Research output: Contribution to journalArticle

Deng, Hua ; Zheng, J. ; Clarke, A. ; Holbrook, J. J. ; Callender, Robert ; Burgner, J. W. / Source of catalysis in the lactate dehydrogenase system. Ground-state interactions in the enzyme·substrate complex. In: Biochemistry. 1994 ; Vol. 33, No. 8. pp. 2297-2305.
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N2 - The Raman spectra of both the NAD-pyruvate and the pyridine aldehyde adenine dinucleotide (PAAD)-pyruvate bound to pig heart, pig muscle, and Bacillus stearothermophilus lactate dehydrogenases were measured and are nearly the same, which is consistent with the conserved shell of residues surrounding the active-site cavity in these enzymes. The symmetrical stretching mode of the pyruvate carboxylate group, found at 1398 cm-1, is shifted only slightly when complexed to these enzymes, which shows that the group remains ionized in the ion pair complex with Arg-171 on the enzyme. The vibrational mode for the carbonyl stretch of the bound pyruvate moiety is shifted about 35 cm-1 to a lower frequency than observed for the carbonyl of unliganded pyruvate in the bacterial enzyme because of polarization of the carbonyl bond. Thus, the bacterial enzyme shows the same substrate activation because of the C+-O- charge separation that was seen previously with the mammalian enzymes. On the basis of an empirical Badger-Bauer relationship between frequency shift and interaction enthalpy, this shift in frequency is equivalent to an approximately -14 to -17 kcal/mol interaction between the enzyme and the adduct C=O coordinate, a substantial part of which is an electrostatic interaction (hydrogen bond) between the C=O and the protonated His-195. Thus, while the C=O bond is polarized on the enzyme (which requires energy), the overall ground-state enthalpy of the carbonyl imidazolium part of the reaction coordinate is stabilized substantially relative to its value in solution, and this is the dominant enthalpic effect on the entire reaction coordinate since the other internal coordinates for the hydride transfer are not much affected during formation of the ternary complex. The total enthalpy of binding for pyruvate analogs to lactate dehydrogenase is nearly the same as the sum of local enthalpies for interactions between pyruvate's C=O and the protein. Thus, even though ligand binding may cause any number of protein conformational changes, the net binding enthalpy of these changes must reflect the sum of a number of large, mostly compensating effects. The Raman spectra of the PAAD-pyruvate adduct bound to two different sets of mutant forms of the bacterial enzyme also were measured. Mutation of Arg-109, which normally hydrogen bonds to the pyruvate C=O, to Gln-109, reduces the extent of C+-O- charge separation by about 4 kcal/mol. Similarly, mutation of Asp- 168, which normally forms an ion pair with His-195 in the presence of a carbonyl-containing substrate to either Asn-168 or Ala-168 also reduces charge separation but to a somewhat greater degree, 4 and 10 kcal/mol, respectively. The ground state for the carbonyl imidazolium interactions of the mutant complexes is thus destabilized relative to the wild-type enzyme, and yet, the height of the transition-state barrier must increase, which clearly indicates the height of the barrier must increase faster than the ground state is destabilized. This view is analyzed from a plot of the log of the hydride transfer step versus the change in frequency of the C=O stretch (or ground-state interaction enthalpy).

AB - The Raman spectra of both the NAD-pyruvate and the pyridine aldehyde adenine dinucleotide (PAAD)-pyruvate bound to pig heart, pig muscle, and Bacillus stearothermophilus lactate dehydrogenases were measured and are nearly the same, which is consistent with the conserved shell of residues surrounding the active-site cavity in these enzymes. The symmetrical stretching mode of the pyruvate carboxylate group, found at 1398 cm-1, is shifted only slightly when complexed to these enzymes, which shows that the group remains ionized in the ion pair complex with Arg-171 on the enzyme. The vibrational mode for the carbonyl stretch of the bound pyruvate moiety is shifted about 35 cm-1 to a lower frequency than observed for the carbonyl of unliganded pyruvate in the bacterial enzyme because of polarization of the carbonyl bond. Thus, the bacterial enzyme shows the same substrate activation because of the C+-O- charge separation that was seen previously with the mammalian enzymes. On the basis of an empirical Badger-Bauer relationship between frequency shift and interaction enthalpy, this shift in frequency is equivalent to an approximately -14 to -17 kcal/mol interaction between the enzyme and the adduct C=O coordinate, a substantial part of which is an electrostatic interaction (hydrogen bond) between the C=O and the protonated His-195. Thus, while the C=O bond is polarized on the enzyme (which requires energy), the overall ground-state enthalpy of the carbonyl imidazolium part of the reaction coordinate is stabilized substantially relative to its value in solution, and this is the dominant enthalpic effect on the entire reaction coordinate since the other internal coordinates for the hydride transfer are not much affected during formation of the ternary complex. The total enthalpy of binding for pyruvate analogs to lactate dehydrogenase is nearly the same as the sum of local enthalpies for interactions between pyruvate's C=O and the protein. Thus, even though ligand binding may cause any number of protein conformational changes, the net binding enthalpy of these changes must reflect the sum of a number of large, mostly compensating effects. The Raman spectra of the PAAD-pyruvate adduct bound to two different sets of mutant forms of the bacterial enzyme also were measured. Mutation of Arg-109, which normally hydrogen bonds to the pyruvate C=O, to Gln-109, reduces the extent of C+-O- charge separation by about 4 kcal/mol. Similarly, mutation of Asp- 168, which normally forms an ion pair with His-195 in the presence of a carbonyl-containing substrate to either Asn-168 or Ala-168 also reduces charge separation but to a somewhat greater degree, 4 and 10 kcal/mol, respectively. The ground state for the carbonyl imidazolium interactions of the mutant complexes is thus destabilized relative to the wild-type enzyme, and yet, the height of the transition-state barrier must increase, which clearly indicates the height of the barrier must increase faster than the ground state is destabilized. This view is analyzed from a plot of the log of the hydride transfer step versus the change in frequency of the C=O stretch (or ground-state interaction enthalpy).

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