Cooperativity in nicotinamide adenine dinucleotide binding induced by mutations of arginine 475 located at the subunit interface in the human liver mitochondrial class 2 aldehyde dehydrogenase

Baoxian Wei, Li Ni, Thomas D. Hurley, Henry Weiner

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Abstract

The low-activity Oriental variant of human mitochondrial aldehyde dehydrogenase possesses a lysine rather than a glutamate at residue 487 in the 500 amino acid homotetrameric enzyme. The glutamate at position 487 formed two salt bonds, one to an arginine at position 264 in the same subunit and the other to arginine 475 in a different subunit [Steinmetz, C. G., Xie, P.-G., Weiner, H., and Hurley, T. D. (1997) Structure 5, 2487-2505]. Mutating arginine 264 to glutamine produced a recombinantly expressed enzyme with nativelike properties; in contrast, mutating arginine 475 to glutamine produced an enzyme that exhibited positive cooperativity in NAD binding. The K(M) for NAD increased 23-fold with a Hill coefficient of 1.8. The binding of both NAD and NADH was affected by the mutation at position 475. Restoring the salt bonds between residues 487 and either or both 264 and 475 did not restore nativelike properties to the Oriental variant. Further, the R475Q mutant was thermally less stable than the native enzyme, Oriental variant, or other mutants. The presence of NAD restored nativelike stability to the mutant. It is concluded that movement of arginine 475 disrupted salt bonds between it and residues other than the one at 487, which caused the apo-R475Q mutant to have properties typical of an enzyme that exhibits positive cooperativity in substrate binding. Breaking the salt bond between glutamate 487 in the Oriental variant and the two arginine residues cannot be the only reason that this enzyme has altered catalytic properties.

Original languageEnglish (US)
Pages (from-to)5295-5302
Number of pages8
JournalBiochemistry
Volume39
Issue number18
DOIs
StatePublished - May 9 2000

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Aldehyde Dehydrogenase
Liver
NAD
Arginine
Mutation
Enzymes
Salts
Glutamic Acid
Glutamine
Lysine
Amino Acids
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Cooperativity in nicotinamide adenine dinucleotide binding induced by mutations of arginine 475 located at the subunit interface in the human liver mitochondrial class 2 aldehyde dehydrogenase",
abstract = "The low-activity Oriental variant of human mitochondrial aldehyde dehydrogenase possesses a lysine rather than a glutamate at residue 487 in the 500 amino acid homotetrameric enzyme. The glutamate at position 487 formed two salt bonds, one to an arginine at position 264 in the same subunit and the other to arginine 475 in a different subunit [Steinmetz, C. G., Xie, P.-G., Weiner, H., and Hurley, T. D. (1997) Structure 5, 2487-2505]. Mutating arginine 264 to glutamine produced a recombinantly expressed enzyme with nativelike properties; in contrast, mutating arginine 475 to glutamine produced an enzyme that exhibited positive cooperativity in NAD binding. The K(M) for NAD increased 23-fold with a Hill coefficient of 1.8. The binding of both NAD and NADH was affected by the mutation at position 475. Restoring the salt bonds between residues 487 and either or both 264 and 475 did not restore nativelike properties to the Oriental variant. Further, the R475Q mutant was thermally less stable than the native enzyme, Oriental variant, or other mutants. The presence of NAD restored nativelike stability to the mutant. It is concluded that movement of arginine 475 disrupted salt bonds between it and residues other than the one at 487, which caused the apo-R475Q mutant to have properties typical of an enzyme that exhibits positive cooperativity in substrate binding. Breaking the salt bond between glutamate 487 in the Oriental variant and the two arginine residues cannot be the only reason that this enzyme has altered catalytic properties.",
author = "Baoxian Wei and Li Ni and Hurley, {Thomas D.} and Henry Weiner",
year = "2000",
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T1 - Cooperativity in nicotinamide adenine dinucleotide binding induced by mutations of arginine 475 located at the subunit interface in the human liver mitochondrial class 2 aldehyde dehydrogenase

AU - Wei, Baoxian

AU - Ni, Li

AU - Hurley, Thomas D.

AU - Weiner, Henry

PY - 2000/5/9

Y1 - 2000/5/9

N2 - The low-activity Oriental variant of human mitochondrial aldehyde dehydrogenase possesses a lysine rather than a glutamate at residue 487 in the 500 amino acid homotetrameric enzyme. The glutamate at position 487 formed two salt bonds, one to an arginine at position 264 in the same subunit and the other to arginine 475 in a different subunit [Steinmetz, C. G., Xie, P.-G., Weiner, H., and Hurley, T. D. (1997) Structure 5, 2487-2505]. Mutating arginine 264 to glutamine produced a recombinantly expressed enzyme with nativelike properties; in contrast, mutating arginine 475 to glutamine produced an enzyme that exhibited positive cooperativity in NAD binding. The K(M) for NAD increased 23-fold with a Hill coefficient of 1.8. The binding of both NAD and NADH was affected by the mutation at position 475. Restoring the salt bonds between residues 487 and either or both 264 and 475 did not restore nativelike properties to the Oriental variant. Further, the R475Q mutant was thermally less stable than the native enzyme, Oriental variant, or other mutants. The presence of NAD restored nativelike stability to the mutant. It is concluded that movement of arginine 475 disrupted salt bonds between it and residues other than the one at 487, which caused the apo-R475Q mutant to have properties typical of an enzyme that exhibits positive cooperativity in substrate binding. Breaking the salt bond between glutamate 487 in the Oriental variant and the two arginine residues cannot be the only reason that this enzyme has altered catalytic properties.

AB - The low-activity Oriental variant of human mitochondrial aldehyde dehydrogenase possesses a lysine rather than a glutamate at residue 487 in the 500 amino acid homotetrameric enzyme. The glutamate at position 487 formed two salt bonds, one to an arginine at position 264 in the same subunit and the other to arginine 475 in a different subunit [Steinmetz, C. G., Xie, P.-G., Weiner, H., and Hurley, T. D. (1997) Structure 5, 2487-2505]. Mutating arginine 264 to glutamine produced a recombinantly expressed enzyme with nativelike properties; in contrast, mutating arginine 475 to glutamine produced an enzyme that exhibited positive cooperativity in NAD binding. The K(M) for NAD increased 23-fold with a Hill coefficient of 1.8. The binding of both NAD and NADH was affected by the mutation at position 475. Restoring the salt bonds between residues 487 and either or both 264 and 475 did not restore nativelike properties to the Oriental variant. Further, the R475Q mutant was thermally less stable than the native enzyme, Oriental variant, or other mutants. The presence of NAD restored nativelike stability to the mutant. It is concluded that movement of arginine 475 disrupted salt bonds between it and residues other than the one at 487, which caused the apo-R475Q mutant to have properties typical of an enzyme that exhibits positive cooperativity in substrate binding. Breaking the salt bond between glutamate 487 in the Oriental variant and the two arginine residues cannot be the only reason that this enzyme has altered catalytic properties.

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