This project, Structural and Function of Biologically Active Molecules, is focussed on activity of three allosteric enzymes: fructose-1,6 bisphosphatase (FBPase) in the gluconeogenic pathway, yeast chorismate mutase (CMase) in the pathway to tyrosine and phenylalanine and aspartate transcarbamylase ATCase in the pyrimidine biosynthetic pathway. FBPase levels are elevated in Type 2 diabetics. Starting from the three-dimensional structures of the T (less active) form and R (more active) forms, attempts are in progress to develop analogues of AMP, the allosteric inhibitor, in order to lower levels of glucose in diabetics. The stages use cycles of synthesis, structure of the enzyme-inhibitor complex by X-ray diffraction, model building and redesign for new synthesis. Inhibitors highly specific for FBPase have been found in the 20nM range for Kd, substantially more strongly bound than AMP (Kd = 1 mu M). Other aspects of structure-function studies of FBPase include establishing the catalytic and regulatory mechanism, especially the roles of the divalent metal cations. Also, as a result of structure determinations active site inhibitors for allosteric (CMase can be designed as herbicides, bacteriocides or fungicides which are harmless to humans who do not make this enzyme. Likewise inhibitors of ATCase, such as PALA (N-phosphonyl-L-aspartate) can be improved upon for control of the pyrimidine pathway in certain cancers. These two regulatory enzymes merit further study, proposed here, in order to understand the structural basis for aspects of allosteric regulation not explainable using the present day models, such as the concerted or sequential models.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM006920-41
Application #
6490010
Study Section
Biochemistry Study Section (BIO)
Program Officer
Flicker, Paula F
Project Start
1975-09-01
Project End
2003-12-31
Budget Start
2002-01-01
Budget End
2002-12-31
Support Year
41
Fiscal Year
2002
Total Cost
$300,090
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

Showing the most recent 10 out of 63 publications