Our objectives are to establish the basis for activity and allosteric regulation of enzymes, using three-dimensional structures as determined by X-ray diffraction methods. Structures are established of complexes of the enzymes with substrate analogues, allosteric effectors, inhibitors, and other molecules and ions, including mutants and other modified forms of the enzymes. The allosteric enzymes under study are fructose-1, 6- bisphosphatase (FBPase), aspartate transcarbamylase (ATCase), and chorismate mutase (CMase) from Saccharomyces cerevisiae. We will also continue studies of the non-allosteric enzyme leucine aminopeptidase (LAP and pepA). The LAP structure at 1.6 angstroms guides us in the two-metal mechanism of FBPase. In the allosteric enzymes, structural studies are continued of binding of various ligands in order to elucidate the mechanisms and pathways for the transmission of conformational information from the allosteric sites to the active sites. Although many such studies have been completed for FBPase and ATCase, the detailed pathways for informational transfer are not yet established. At this early stage both T and R structures are known for allosteric CMase. The structural basis for design of an analogue of the allosteric inhibitor (AMP) of FBPase is being established in order to develop a drug for treatment of Type II diabetes. A highly specific analogue (K(d)=0.1 microM) of AMP (K(d)=1 microM) has been obtained as an inhibitor for FBPase, an enzyme which is elevated in Type II diabetes; the method consisted of repeated consecutive cycles of structures of FBPase bound to the inhibitor followed by model building of a newly designed inhibitor from the X-ray results, followed by chemical synthesis. Active site inhibitors can be designed from structures for ATCase (control pyrimidine synthesis), CMase (to make herbicides, bacteriocides or fungicides), and leucine aminopeptidase (to control protein degradation and hormone processing).

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM006920-38
Application #
2684609
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1975-09-01
Project End
1999-12-31
Budget Start
1998-04-01
Budget End
1999-12-31
Support Year
38
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Harvard University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
071723621
City
Cambridge
State
MA
Country
United States
Zip Code
02138
Lipscomb, William N; Kantrowitz, Evan R (2012) Structure and mechanisms of Escherichia coli aspartate transcarbamoylase. Acc Chem Res 45:444-53
Lipscomb, W N (1994) Aspartate transcarbamylase from Escherichia coli: activity and regulation. Adv Enzymol Relat Areas Mol Biol 68:67-151
Xue, Y; Huang, S; Liang, J Y et al. (1994) Crystal structure of fructose-1,6-bisphosphatase complexed with fructose 2,6-bisphosphate, AMP, and Zn2+ at 2.0-A resolution: aspects of synergism between inhibitors. Proc Natl Acad Sci U S A 91:12482-6
Chook, Y M; Gray, J V; Ke, H et al. (1994) The monofunctional chorismate mutase from Bacillus subtilis. Structure determination of chorismate mutase and its complexes with a transition state analog and prephenate, and implications for the mechanism of the enzymatic reaction. J Mol Biol 240:476-500
Zhang, Y; Liang, J Y; Huang, S et al. (1994) Toward a mechanism for the allosteric transition of pig kidney fructose-1,6-bisphosphatase. J Mol Biol 244:609-24
Reinisch, K M; Chen, L; Verdine, G L et al. (1994) Crystallization and preliminary crystallographic analysis of a DNA (cytosine-5)-methyltransferase from Haemophilus aegyptius bound covalently to DNA. J Mol Biol 238:626-9
Kim, H; Lipscomb, W N (1994) Structure and mechanism of bovine lens leucine aminopeptidase. Adv Enzymol Relat Areas Mol Biol 68:153-213
Xue, Y; Lipscomb, W N (1994) The crystallization and preliminary X-ray analysis of allosteric chorismate mutase. J Mol Biol 241:273-4
Gidh-Jain, M; Zhang, Y; van Poelje, P D et al. (1994) The allosteric site of human liver fructose-1,6-bisphosphatase. Analysis of six AMP site mutants based on the crystal structure. J Biol Chem 269:27732-8
Xue, Y; Lipscomb, W N; Graf, R et al. (1994) The crystal structure of allosteric chorismate mutase at 2.2-A resolution. Proc Natl Acad Sci U S A 91:10814-8

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