Description

Interfaces between subunits play important roles in communicating activation or inhibition critical for an organism?s survival. The molecular mechanism of such interactions has not been fully understood. Glyoxysomal malate dehydrogenase (gMDH) is a homodimeric enzyme whose interface is thought to participate in regulation both by citrate and by substrate inhibition and a proposed reciprocating subunit mechanism of the overall catalytic cycle. The interface contains a variety of electrostatic and non-polar interactions. To understand the mechanism of subunit interactions we have probed the role of individual sidechains at the interface by site directed mutagenesis to determine the effects on stability, activity, and regulation. L269 and S266 appear to form a relay system that connects the active site to the interface. We have constructed, expressed, and purified two mutants, L269A and S266A. Biochemical and biophysical techniques were used to evaluate the structure and stability of the mutants. Size exclusion chromatography and chemical cross-linking experiments demonstrate that L269A and S266A are dimeric. Comparison of CD spectra of the mutants to the native showed similar secondary structure and, via thermal melts, TM values. To study the role of these residues in gMDH activity, initial rate kinetic studies were performed and showed that S266A and L269A have a lower affinity for OAA while, unlike the native enzyme, the L269A mutant is not saturated by NADH over experimental ranges. This, coupled with the decreased activity of both mutants, suggests that the residues play a key role in the binding of NADH and regulation of gMDH by OAA. This was supported by fluorescence based thermal shift (FTS) studies that demonstrated how NADH binding is greatly decreased in L269A. The results suggest that subunit interactions mediated at least in part by S266 and L269 are part of a relay system between active sites in the homodimer that regulates activity and inhibition of gMDH

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Probing the role of the interface on activity and regulation of gMDH

Interfaces between subunits play important roles in communicating activation or inhibition critical for an organism?s survival. The molecular mechanism of such interactions has not been fully understood. Glyoxysomal malate dehydrogenase (gMDH) is a homodimeric enzyme whose interface is thought to participate in regulation both by citrate and by substrate inhibition and a proposed reciprocating subunit mechanism of the overall catalytic cycle. The interface contains a variety of electrostatic and non-polar interactions. To understand the mechanism of subunit interactions we have probed the role of individual sidechains at the interface by site directed mutagenesis to determine the effects on stability, activity, and regulation. L269 and S266 appear to form a relay system that connects the active site to the interface. We have constructed, expressed, and purified two mutants, L269A and S266A. Biochemical and biophysical techniques were used to evaluate the structure and stability of the mutants. Size exclusion chromatography and chemical cross-linking experiments demonstrate that L269A and S266A are dimeric. Comparison of CD spectra of the mutants to the native showed similar secondary structure and, via thermal melts, TM values. To study the role of these residues in gMDH activity, initial rate kinetic studies were performed and showed that S266A and L269A have a lower affinity for OAA while, unlike the native enzyme, the L269A mutant is not saturated by NADH over experimental ranges. This, coupled with the decreased activity of both mutants, suggests that the residues play a key role in the binding of NADH and regulation of gMDH by OAA. This was supported by fluorescence based thermal shift (FTS) studies that demonstrated how NADH binding is greatly decreased in L269A. The results suggest that subunit interactions mediated at least in part by S266 and L269 are part of a relay system between active sites in the homodimer that regulates activity and inhibition of gMDH

 

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