Molecular properties of p-(dimethylamino)benzaldehyde bound to liver alcohol dehydrogenase

A Raman spectroscopic study

Robert Callender, Dehuai Chen, Johan Lugtenburg, Charlotte Martin, Kee Woo Rhee, Donald Sloan, Robert Vandersteen, Kwok To Yue

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

We have studied the binding nature of an aromatic aldehyde to the catalytic site of liver alcohol dehydrogenase from horse (LADH) using preresonance Raman spectroscopy. The compound p-(dimethylamino)benzaldehyde (DABA) is converted to the corresponding alcohol in the presence of nicotinamide adenine dinucleotide (NADH) and a catalytic amount of enzyme at neutral pH. A stable ternary complex of LADH/NADH/DABA can be formed if enzyme and coenzyme are in excess at high pH [Jagodzinski, P. W., Funk, G. F., & Peticolas, W. L. (1982) Biochemistry 21, 2193-2202]. We have obtained the preresonance Raman spectrum of bound DABA by subtracting the contribution of the binary complex of LADH/NADH from the spectrum of this stable ternary complex. In order to understand the normal mode patterns of DABA, four isotopically labeled DABA derivatives were synthesized and their Raman spectra, in solution and in the ternary complex, were measured. Three of these compounds contain substitutions in the functionally important aldehyde moiety: (i) In one such substitution, the aldehydic hydrogen atom was replaced by a deuterium; (ii) in another, this hydrogen atom was replaced by deuterium, and the aldehydic carbon atom was replaced by 13C; and (iii) in the third derivative, only the carbon atom was replaced by 13C. The fourth derivative has had the two hydrogen atoms at the 3- and 5-positions of the DABA ring replaced by deuterium atoms. We find that many of the spectral modes are fairly extended, involving both stretching and bending motions of the entire molecule, although a few modes are quite localized. We find that the normal mode structure of DABA changes considerably when it binds to LADH/NADH. As a model for the bound DABA, we have examined the zinc complexes of DABA (and all four isotopically labeled samples) in anhydrous diethyl ether and methylene chloride. A striking correspondence between the Raman spectra of the enzyme-bound DABA and DABA-Zn complexes in solution is found, which extends to all the isotopically labeled derivatives. This suggests that one of the major roles of LADH in the binding of DABA is to provide a divalent zinc ion to form a first-sphere Lewis acid complex. The data also suggest other interactions between enzyme-bound DABA with its protein surroundings and with the coenzyme NADH are quite minor. An estimate of the carbonyl bond character of bound DABA had been made on the basis of the response of Raman bands to isotopic labeling and on trends observed in spectra of DABA in solvents of various polarities. It is found to have a bond order between a single and double bond. We discuss a possible role of the zinc ion at the enzyme's active site and subsequently the mechanism of the enzymatic function of LADH in view of these results.

Original languageEnglish (US)
Pages (from-to)3672-3681
Number of pages10
JournalBiochemistry
Volume27
Issue number10
StatePublished - 1988
Externally publishedYes

Fingerprint

Alcohol Dehydrogenase
Liver
NAD
Horses
Atoms
Deuterium
Derivatives
Raman scattering
Zinc
Hydrogen
benzaldehyde
Enzymes
Aldehydes
Catalytic Domain
Substitution reactions
Carbon
Enzymes and Coenzymes
Ions
Lewis Acids
Biochemistry

ASJC Scopus subject areas

  • Biochemistry

Cite this

Callender, R., Chen, D., Lugtenburg, J., Martin, C., Rhee, K. W., Sloan, D., ... Yue, K. T. (1988). Molecular properties of p-(dimethylamino)benzaldehyde bound to liver alcohol dehydrogenase: A Raman spectroscopic study. Biochemistry, 27(10), 3672-3681.

Molecular properties of p-(dimethylamino)benzaldehyde bound to liver alcohol dehydrogenase : A Raman spectroscopic study. / Callender, Robert; Chen, Dehuai; Lugtenburg, Johan; Martin, Charlotte; Rhee, Kee Woo; Sloan, Donald; Vandersteen, Robert; Yue, Kwok To.

In: Biochemistry, Vol. 27, No. 10, 1988, p. 3672-3681.

Research output: Contribution to journalArticle

Callender, R, Chen, D, Lugtenburg, J, Martin, C, Rhee, KW, Sloan, D, Vandersteen, R & Yue, KT 1988, 'Molecular properties of p-(dimethylamino)benzaldehyde bound to liver alcohol dehydrogenase: A Raman spectroscopic study', Biochemistry, vol. 27, no. 10, pp. 3672-3681.
Callender, Robert ; Chen, Dehuai ; Lugtenburg, Johan ; Martin, Charlotte ; Rhee, Kee Woo ; Sloan, Donald ; Vandersteen, Robert ; Yue, Kwok To. / Molecular properties of p-(dimethylamino)benzaldehyde bound to liver alcohol dehydrogenase : A Raman spectroscopic study. In: Biochemistry. 1988 ; Vol. 27, No. 10. pp. 3672-3681.
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T1 - Molecular properties of p-(dimethylamino)benzaldehyde bound to liver alcohol dehydrogenase

T2 - A Raman spectroscopic study

AU - Callender, Robert

AU - Chen, Dehuai

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AU - Sloan, Donald

AU - Vandersteen, Robert

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N2 - We have studied the binding nature of an aromatic aldehyde to the catalytic site of liver alcohol dehydrogenase from horse (LADH) using preresonance Raman spectroscopy. The compound p-(dimethylamino)benzaldehyde (DABA) is converted to the corresponding alcohol in the presence of nicotinamide adenine dinucleotide (NADH) and a catalytic amount of enzyme at neutral pH. A stable ternary complex of LADH/NADH/DABA can be formed if enzyme and coenzyme are in excess at high pH [Jagodzinski, P. W., Funk, G. F., & Peticolas, W. L. (1982) Biochemistry 21, 2193-2202]. We have obtained the preresonance Raman spectrum of bound DABA by subtracting the contribution of the binary complex of LADH/NADH from the spectrum of this stable ternary complex. In order to understand the normal mode patterns of DABA, four isotopically labeled DABA derivatives were synthesized and their Raman spectra, in solution and in the ternary complex, were measured. Three of these compounds contain substitutions in the functionally important aldehyde moiety: (i) In one such substitution, the aldehydic hydrogen atom was replaced by a deuterium; (ii) in another, this hydrogen atom was replaced by deuterium, and the aldehydic carbon atom was replaced by 13C; and (iii) in the third derivative, only the carbon atom was replaced by 13C. The fourth derivative has had the two hydrogen atoms at the 3- and 5-positions of the DABA ring replaced by deuterium atoms. We find that many of the spectral modes are fairly extended, involving both stretching and bending motions of the entire molecule, although a few modes are quite localized. We find that the normal mode structure of DABA changes considerably when it binds to LADH/NADH. As a model for the bound DABA, we have examined the zinc complexes of DABA (and all four isotopically labeled samples) in anhydrous diethyl ether and methylene chloride. A striking correspondence between the Raman spectra of the enzyme-bound DABA and DABA-Zn complexes in solution is found, which extends to all the isotopically labeled derivatives. This suggests that one of the major roles of LADH in the binding of DABA is to provide a divalent zinc ion to form a first-sphere Lewis acid complex. The data also suggest other interactions between enzyme-bound DABA with its protein surroundings and with the coenzyme NADH are quite minor. An estimate of the carbonyl bond character of bound DABA had been made on the basis of the response of Raman bands to isotopic labeling and on trends observed in spectra of DABA in solvents of various polarities. It is found to have a bond order between a single and double bond. We discuss a possible role of the zinc ion at the enzyme's active site and subsequently the mechanism of the enzymatic function of LADH in view of these results.

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