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 o f identical subunits. In the crystal structures of 2 prokaryotic and 2 eukaryotic forms, the subunit interface is conformationally homologou s. To determine whether or not the quaternary structure of MDH is link ed to the catalytic activity, mutant forms of the enzyme from Escheric hia coli have been constructed. Utilizing the high-resolution structur e of E. coli MDH, the dimer interface was analyzed critically for side chains that were spatially constricted and needed for electrostatic i nteractions. Two such residues were found, D45 and S226. At their near est point in the homodimer, they are in different subunits, hydrogen b ond across the interface, and do not interact with any catalytic resid ues. Each residue was mutated to a tyrosine, which should disrupt the interface because of its large size. All mutants were cloned and purif ied to homogeneity from an mdh(-) E. coli strain (BHB111). Gel filtrat ion of the mutants show that D45Y and D45Y/S226Y are both monomers, wh ereas the S226Y mutant remains a dimer. The monomeric D45Y and D45Y/S2 26Y mutants have 14,000- and 17,500-fold less specific activity, respe ctively, than the native enzyme. The dimeric S226Y has only 1.4-fold l ess specific activity. All forms crystallized, indicating they were no t random coils. Data have been collected to 2.8 Angstrom resolution fo r the D45Y mutant. The mutant is not isomorphous with the native prote in and work is underway to solve the structure by molecular replacemen t.