Engineering the quaternary structure of an enzyme: construction and analysis of a monomeric form of malate dehydrogenase from Escherichia coli.
The citric acid cycle enzyme, malate dehydrogenase (MDH), is a dimer of identical subunits. In the crystal structures of 2 prokaryotic and 2 eukaryotic forms, the subunit interface is conformationally homologous. To determine whether or not the quaternary structure of MDH is linked to the catalytic activity, mutant forms of the enzyme from Escherichia coli have been constructed. Utilizing the high-resolution structure of E. coli MDH, the dimer interface was analyzed critically for side chains that were spatially constricted and needed for electrostatic interactions. Two such residues were found, D45 and S226. At their nearest point in the homodimer, they are in different subunits, hydrogen bond across the interface, and do not interact with any catalytic residues. Each residue was mutated to a tyrosine, which should disrupt the interface because of its large size. All mutants were cloned and purified to homogeneity from an mdh- E. coli strain (BHB111). Gel filtration of the mutants show that D45Y and D45Y/S226Y are both monomers, whereas the S226Y mutant remains a dimer. The monomeric D45Y and D45Y/S226Y mutants have 14,000- and 17,500-fold less specific activity, respectively, than the native enzyme. The dimeric S226Y has only 1.4-fold less specific activity. All forms crystallized, indicating they were not random coils. Data have been collected to 2.8 A resolution for the D45Y mutant. The mutant is not isomorphous with the native protein and work is underway to solve the structure by molecular replacement.
Breiter
D R
DR
Department of Biochemistry, University of Minnesota, Minneapolis 55455.
Resnik
E
E
Banaszak
L J
LJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D011487:Protein Conformation; D015202:Protein Engineering
D008291:Malate Dehydrogenase