1999 Milwaukee Section Award Evolution of an Enzyme Active Site: The Three-dimensional Structures of Acyl-COA Dehydrogenases Jung-Ja Park Kim Department of Biochemistry Medical College of Wisconsin

Friday November 19, 1999 Teaching, Learning and Technology Center Alverno College 3400 South 43rd Street Milwaukee, WI

DIRECTIONS

Fatty acid oxidation is the principal energy-yielding process in virtually all mammalian tissues, including the liver and heart. It is carried out by a series of enzymes that successively cleave acetyl-CoA fragments from fatty acyl-CoA, which is derived from dietary fat. The initial reaction in each cycle of fatty acid oxidation is the dehydrogenation of a fatty acyl-CoA to the corresponding trans-2,3-enoyl-CoA. The reaction is catalyzed by a family of related enzymes, acyl-CoA dehydrogenases (ACDs), that contain flavin adenine dinucleotide (FAD, a vitamin B2 derivative).

The family includes four ACD enzymes that exhibit distinct but overlapping chain length specificities for straight-chain acyl-CoA thioesters. In addition, there are three branched-chain specific ACDs that are involved in amino acid catabolism. Except for the membrane-bound very long chain acyl-CoA dehydrogenase, these enzymes are very similar in the chemistry of their catalytic action and their molecular properties. Amino acid sequence analyses reveal 30-35% identity, suggesting that they are evolved from a common ancestral gene. Furthermore, the three-dimensional structures of several ACDs show that their overall polypeptide folding and the active site geometry are very similar, confirming their common ancestry. However, a close examination of the active site in each of the known three-dimensional structures of the ACD members reveals that the shape and size of the substrate binding cavities differ in order to accommodate specific substrates. Furthermore, Glutamate 376, identified as the catalytic base in medium- and short-chain ACDs is not conserved in long-chain acyl-CoA dehydrogenase and isovaleryl-CoA dehydrogenase, although the sequence identity is retained throughout the lengths of their entire polypeptide chains. In the latter enzymes, the catalytic residue was suggested to be Glu261 by molecular modeling and mutagenesis and was later confirmed by crystallographic analysis. The two glutamates [376 (medium chain-ACD) and 261 (long chain-ACD)], separated by over 100 residues in the linear amino acid sequence, are spatially contiguous in the active site, approaching from different directions to the proton to be abstracted. The distance from the substrate and angle at which the catalytic glutamate approaches the substrate, together with the size and shape of the active site cavity, modulate the reaction rate and determine the substrate specificity in each of the ACD family members.

Medium chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common among fatty acid metabolic disorders. The three-dimensional structure of the enzyme reveals the structural basis for the low activity of the mutant enzyme found in patients with the disorder.

Jung-Ja Park Kim, Ph.D., is a Professor of Biochemistry at the Medical College of Wisconsin, Milwaukee, WI. She received her B.S. in Chemistry from Seoul, Korea in 1963. She earned her Ph.D. at Cornell University, Ithaca, N.Y., in 1969 doing X-ray structural work, followed by a Postdoctoral Appointment at McMaster University, Hamilton, Ontario. A second Postdoctoral Fellow and Research Scientist Appointment at Massachusetts Institute of Technology led to Dr. Kim's solving of the three dimensional structure of Transfer-RNA, an important component in protein synthesis.

Much of Dr. Kim's work at the Medical College has involved solving the X-ray structures of the flavin containing enzymes in the fatty acid oxidation pathway called Acyl-CoA Dehydrogenases and Electron Transfer Factors. This work can be found in several basic biochemistry texts, VOET and VOET and Garrett and Grisham. Recently she solved the X-ray structure of NADPH-Cytochrome P450 Reductase, part of the important oxygenating and hydroxylating systems found in nature.

Importantly, she has sought and received funding for bringing an X-ray Diffractometer for Macromolecular Structure Analysis into the Milwaukee area, along with an Area Detector Rotating Anode for Protein Crystallography and a Computer Graphics System for Macromolecular Structure Analysis. Her willingness to collaborate with colleagues has resulted in a number of additional new structures, Cation-dependent Mannose 6-phosphate Receptor, Phosphoribulokinase, and Ubiquitin-Cross Reacting Protein.

Dr. Jung Ja Kim, who has over sixty publications, has been invited to speak at a number of International Symposia on Flavins and Flavoproteins and Cytochrome P450 Proteins. She has written a number of invited reviews and symposia publications. Currently Dr. Kim is working with a consortium of Midwest Universities for a synchrotron beam access station at the Advanced Photon Source at Argonne National Laboratories. Such a resource will improve the resolution of enzyme and protein electron density maps, so that protein structure and enzyme catalysis may be more definitively understood. Further much smaller protein crystals will become useful in X-ray structural work.