Crystal Studies of Enolase and Transcarboxylase

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Structure function relationship in enolase will be studied using a combination of x-ray crystallography, site directed mutagenesis, and kinetic studies. There are two, or perhaps three, divalent metal ion binding sites per subunit of enolase which are sequentially filled. Some metal ions activate enolase, others bind strongly but don not activate. Dr. Lebioda will study ternary and quaternary complexes of enolase- metal ion-substrate-metal ion to image the sequential steps in the enzyme mechanism. Crystals of mutant enolase will be obtained (in collaboration with Drs. Brewer and Robson) and their structure determined. The mechanism of enolase inhibition by fluoride ions will be studied. Interaction of single stranded DNA with enolase will be modeled by studying the complexes of oligonucleotides with enolase. The yeast enolase crystals scatter to 1.8A resolution; this makes the system an excellent object of the proposed studies. In addition to enolase research, Dr. Lebioda also proposes to study the structure of another metalloenzyme, transcarboxylase (TC). There is significant sequence identity indicating homology between TC and one or more domains of other biotin enzymes. Thus, its structure will be representative for this class of proteins.