TY - JOUR
T1 - A Vibrational Analysis of the Catalytically Important C4-H Bonds of NADH Bound to Lactate or Malate Dehydrogenase
T2 - Ground-State Effects
AU - Deng, Hua
AU - Zheng, Jie
AU - Sloan, Donald
AU - Burgner, John
AU - Callendcr, Robert
PY - 1992/2/1
Y1 - 1992/2/1
N2 - We have measured the frequency of the carbon-hydrogen stretching mode of the pro-R and pro-S C4-H bonds of NADH in solution and when bound to pig heart lactate (LDH) or mitochondrial malate (mMDH) dehydrogenases. This is achieved by specifically deuterating the C4 pro-R or pro-S hydrogens of NADH and determining the frequencies of the resulting C4-D stretches by Raman difference spectroscopy. We find that the frequencies of the two C4-D stretching modes for the two bonds are essentially the same for the unliganded coenzyme. On the other hand, the position of the pro-S-[4-2H]NADH stretch shifts upward by about 23-30 cm-1 in its binary complex with either lactate or malate dehydrogenase relative to that observed in solution, while that for the bound pro-R[4-2H]NADH is relatively unchanged. The fact that the frequency of the pro-R hydrogen is not significantly affected during complex formation suggests that the rale enhancements for reaction of substrate with NADH brought about by both pig heart LDH and mMDH apparently do not involve either stabilization or destabilization of the pro-R hydrogen of NADH in enzyme-coenzyme binary complexes, in agreement with previous chemical studies. That these proteins are able to regulate the frequencies of the two C4-D bonds differentially, and hence the electronic distributions in these bonds, has important implications for the stereochemical reactions catalyzed by the NAD dehydrogenases, and this is discussed. We have studied a number of factors which can affect the C4-H stretch frequency by normal mode analyses of our Raman results based on semiempirical quantum mechanical calculations (MINDO/3, MNDO, and AMI). These factors include the interaction between the nicotinamide ring nitrogen and the ribose oxygen, the torsional angle of the amide arm, puckering of the ring, and the external charge or dipole modeled by a formaldehyde. Within the range of our study, the positions of the C4-D stretches may be understood as the result of two conformational changes of the nicotinamide ring that occur when NADH forms a binary complex with LDH or mMDH: the rotation of the amide group from a solution syn to anti in situ and the adoption of a “half-boat” of the dihydronicotinamide ring of NADH when bound to the two enzymes from an essentially planar solution structure. The estimated angle of the C4 ring carbon with respect to the other carbon atoms is around 15°, with the pro-R hydrogen at a pseudoaxial position and the pro-S hydrogen at a pseudoequatorial position. Our calculations also show that electrostatic interactions, as modeled by the interaction between the C-D bond and a point charge or a carbonyl dipole, can also be important in determining the C-D stretch frequency and differences between the pro-R and pro-S bond frequencies, although they apparently have no major effect in LDH or mMDH.
AB - We have measured the frequency of the carbon-hydrogen stretching mode of the pro-R and pro-S C4-H bonds of NADH in solution and when bound to pig heart lactate (LDH) or mitochondrial malate (mMDH) dehydrogenases. This is achieved by specifically deuterating the C4 pro-R or pro-S hydrogens of NADH and determining the frequencies of the resulting C4-D stretches by Raman difference spectroscopy. We find that the frequencies of the two C4-D stretching modes for the two bonds are essentially the same for the unliganded coenzyme. On the other hand, the position of the pro-S-[4-2H]NADH stretch shifts upward by about 23-30 cm-1 in its binary complex with either lactate or malate dehydrogenase relative to that observed in solution, while that for the bound pro-R[4-2H]NADH is relatively unchanged. The fact that the frequency of the pro-R hydrogen is not significantly affected during complex formation suggests that the rale enhancements for reaction of substrate with NADH brought about by both pig heart LDH and mMDH apparently do not involve either stabilization or destabilization of the pro-R hydrogen of NADH in enzyme-coenzyme binary complexes, in agreement with previous chemical studies. That these proteins are able to regulate the frequencies of the two C4-D bonds differentially, and hence the electronic distributions in these bonds, has important implications for the stereochemical reactions catalyzed by the NAD dehydrogenases, and this is discussed. We have studied a number of factors which can affect the C4-H stretch frequency by normal mode analyses of our Raman results based on semiempirical quantum mechanical calculations (MINDO/3, MNDO, and AMI). These factors include the interaction between the nicotinamide ring nitrogen and the ribose oxygen, the torsional angle of the amide arm, puckering of the ring, and the external charge or dipole modeled by a formaldehyde. Within the range of our study, the positions of the C4-D stretches may be understood as the result of two conformational changes of the nicotinamide ring that occur when NADH forms a binary complex with LDH or mMDH: the rotation of the amide group from a solution syn to anti in situ and the adoption of a “half-boat” of the dihydronicotinamide ring of NADH when bound to the two enzymes from an essentially planar solution structure. The estimated angle of the C4 ring carbon with respect to the other carbon atoms is around 15°, with the pro-R hydrogen at a pseudoaxial position and the pro-S hydrogen at a pseudoequatorial position. Our calculations also show that electrostatic interactions, as modeled by the interaction between the C-D bond and a point charge or a carbonyl dipole, can also be important in determining the C-D stretch frequency and differences between the pro-R and pro-S bond frequencies, although they apparently have no major effect in LDH or mMDH.
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U2 - 10.1021/bi00136a022
DO - 10.1021/bi00136a022
M3 - Article
C2 - 1599930
AN - SCOPUS:0026755601
SN - 0006-2960
VL - 31
SP - 5085
EP - 5092
JO - Biochemistry
JF - Biochemistry
IS - 21
ER -