Determinants of substrate recognition and mechanisms of subunit exchange

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2007-01-01
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Zhou, Yang
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Richard B. Honzatko
Herbert J. Fromm
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Biochemistry, Biophysics and Molecular Biology

The Department of Biochemistry, Biophysics, and Molecular Biology was founded to give students an understanding of life principles through the understanding of chemical and physical principles. Among these principles are frontiers of biotechnology such as metabolic networking, the structure of hormones and proteins, genomics, and the like.

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The Department of Biochemistry and Biophysics was founded in 1959, and was administered by the College of Sciences and Humanities (later, College of Liberal Arts & Sciences). In 1979 it became co-administered by the Department of Agriculture (later, College of Agriculture and Life Sciences). In 1998 its name changed to the Department of Biochemistry, Biophysics, and Molecular Biology.

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1959–present

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  • Department of Biochemistry and Biophysics (1959–1998)

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Abstract

Adenylosuccinate synthetase (ADSS) offers a good opportunity to measure the effect of non-bonded interactions. Both IMP and 2'-deoxy-IMP are good substrates supporting identical binding affinities and maximal velocities; however, L-aspartate exhibits significant differences in binding affinities depending on which nucleotide is used as a substrate. Crystal structures of both complexes were identical except for the absence of the 2'-OH group in the dexoy-nucleotide complex. The decrease in energy (∼2 kcal/mole) in non-bonded interactions due to the loss of one atom explains the nearly 40-fold difference in the binding affinity of L-aspartate and an analog of L-aspartate called hadacidin. Single-atom cavities can profoundly influence the binding affinity of a ligand to a protein or the binding of two proteins to each other.;Sequence position 273 in Escherichia coli adenylosuccinate synthetase has a significant influence on the recognition of one of its substrates L-aspartate. The mutation of Valine273 to threonine, alanine, or asparagine causes 15-40 fold changes in the K m for L-asparate. Crystallizing these mutant proteins with and without ligands, and subsequent crystallographic investigations, revealed the basis for differences in Km values.;FBPase catalyzes the formation of fructose 1,6-bisphosphate to fructose 1,6-bisphosphate and inorganic phosphate. It exists in at least two quaternary states called R and T. Active site ligands such as Fru-6-P, Fru-1,6-P 2, and Fru-2,6-P2 can limit certain subunit exchange reactions, and AMP can abolish exchange altogether. In total, a mixture of two distinguishable FBPases leads to seven different tetramers. By locating the probes at specific positions, the separation of tetramer into dimers, and dimers into monomer, could be monitored independently. Furthermore, by quantifying the time-dependent change in fluorescence the elementary rate constants for forward and reverse reactions in tetramer/dimer and dimer/monomer equilibriums could be determined. These constants allowed the simulation of subunit exchange reactions in agreement with observed fluorescence changes. The approach here is innovative, broadly applicable to any multisubunit system and easier to accomplish than comparable fluorescence resonance energy transfer experiments.

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Mon Jan 01 00:00:00 UTC 2007