RUI: Structure and Function in Enzymes Adapted to Extreme Cold

Franklin And Marshall College, Lancaster PA

Investigators

Abstract

Enzymes exposed to novel thermal environments undergo selection to maintain functional parameters, such as catalytic rate and substrate affinity, at physiologically appropriate levels. The amount of structural modification necessary to produce these compensatory functional changes is unclear, but it has been hypothesized that temperature adaptation in enzymes takes place via changes in flexibility in or near structures that move during catalysis. The objective of the research is to test this hypothesis, focusing on the following questions: As the thermal environment changes, how much structural modification is necessary to maintain optimal enzyme function? Do orthologous enzymes from different species adapted to the same thermal environment show similar changes in catalytic function, and in underlying structure? Do unrelated enzymes show analogous changes in structure in response to temperature change - specifically, do temperature-adaptive changes occur in areas associated with movement during catalysis? To answer these questions, three glycolytic enzymes will be examined - muscle-type lactate dehydrogenase, pyruvate kinase and aldolase - from two unrelated groups of marine fishes adapted to extreme cold - the Antarctic notothenioids and the Arctic and Polar cods. The project will be divided into four segments: First, for each ortholog, the kinetic parameters kcat (catalytic rate) and Km (substrate affinity) will be determined across a range of temperatures. Second, the primary structure of each enzyme will be deduced from complementary DNA sequences. Third, computer models of the three-dimensional structures of each of the polar orthologs will be created, in order to determine which amino acid substitutions shared by the polar enzyme orthologs are most likely to affect enzyme function in a temperature-adaptive manner. Fourth, representative polar orthologs will be modified through site-directed mutagenesis to confirm the role of those residues important in cold adaptation. Km and kcat values of the mutants will be determined to test the hypothesis that temperature-adaptive structural changes occur in areas undergoing catalytically important conformational changes. Organisms adapt to long-term changes in temperature at a variety of levels, from alterations in body shape and size to modifications in the functioning of biochemical pathways. This project is designed to determine how changes in the structure of enzymes - proteins that act as catalysts in biochemical processes - lead to optimal enzyme function at extremely cold temperatures. Common metabolic enzymes from Antarctic and Arctic fishes will be compared to discover whether the same types of functional adaptation have occurred in these unrelated species. An important goal of this research is to find those amino acid substitutions (changes in the basic building blocks of the proteins) responsible for cold adaptation, and to determine whether similar substitutions have occurred in the enzymes of the different fish species. The research will help clarify the rate and extent of molecular evolution as organisms adapt to novel environments.