One major area of reseach in my laboratory is the study of the regulation of the rates of metabolic pathways by allosteric enzymes. Allosteric control is an extremely important mechanism for cellular regulation.  Allosteric enzymes play a pivotal role in cells because they have two functions – they not only catalyze reactions in metabolic pathways, but also control the rates of these pathways.  Regulation by allosteric enzymes involves the binding of signaling molecules to specific regulatory sites, and this binding induces conformational changes which result in alterations in catalytic activity. 

Our work has concentrated on E. coli aspartate transcarbamoylase (ATCase), the enzyme that catalyzes the committed step in pyrimidine nucleotide biosynthesis: the reaction between carbamoyl phosphate and L-aspartate to form N-carbamoyl-L-aspartate and inorganic phosphate.

In addition to catalysis of the above reaction ATCase also regulates pyrimidine nucleotide biosynthesis. CTP and CTP plus UTP, the end products of pyrimidine nucleotide biosynthesis inhibit the enzyme, while ATP, the product of the parallel purine nucleotide pathway activates the enzyme. The enzymatic control exerted by ATCase helps to balance the pools of purine and pyrimidine nucleotides in the cell. Regulation occurs by the binding of the effector molecules at an allosteric site, remove from the active site, and including conformational changes in the enzyme. E. coli ATCase, is composed of 12 polypeptide chains, six larger chains that contain the active sites and six smaller chains that contain the regulatory binding sites. Two distinct conformations of thye enzyme have been identified, the T or tense form, which has low activity and low affinity, and the R or relaxed form, which has high activity and high affinity. The enzyme undergoes an amazing conformational change from the T to the R state that can be monitored by small-angle x-ray scattering in solution as well a X-ray crystallography.

The second allosteric enzyme being studied is fructose 1,6-bisphosphatase (FBPase), an enzyme that is critically involved in the control of gluconeogenesis and is a target for anti-diabetic drugs.  In diabetes fructose 1,6-bisphosphatase fails to regulate gluconeogenesis resulting in altered blood sugar levels. FBPase is a homotetramer catalyzing the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate.

FBPase is allosterically inhibited by AMP and fructose 2,6-bisphosphate, however, as opposed to ATCase the catalytic and regulatory binding sites are on same polypeptide chain. For FBPase the R to T conformational change involves a rotation of the two dimeric halves of the molecule with respect to each other by about 17°. These quaternary changes are accompanied by alterations in the tertiary structure, the most significant of which involve large move­ments of loops. The allosteric transition from the T to the R state results in the formation of the catalytically competent active site mainly due to the rearrangement of a loop that brings a metal binding site in close proximity to the active site.

The control of metabolic pathways by allosteric enzymes is analogous to a molecular value which has “on” and “off” positions.  In the "off" position flow through the pathway is severely hindered, while in the “on” position the flow is normal.  Koshland has recently said that “this type of cooperativity, which has since been shown in many enzymes, receptors, and ion channels, is of critical importance to both evolution and the field of proteomics because it can serve as a general model for the way in which the networks of interacting enzymes of metabolic pathways are regulated.”  For a comprehensive understanding of allosteric regulation we are attempting to answer three fundamental questions: (i) What is the mechanism by which the enzyme accelerates the reaction rate? (ii) What is the mechanism by which the enzyme shifts between the “off” and “on” states? and (iii) What is the manner by which the activity of the enzyme is altered by the binding of regulatory molecules.

Important References:

Kantrowitz, E. R. and Lipscomb, W. N. (1990) "Escherichia coli Aspartate Transcarbamylase:  The Molecular Basis for a Concerted Allosteric Transition, TrendsBiochem. Sci.,15, 53-59.

Jin, L., Stec, B., Lipscomb, W. N., and Kantrowitz, E. R. (1999) "Insights into the mechanism of catalysis and heterotropic regulation of E. coli aspartate transcarbamoylase based upon a structure of  enzyme complexed with the bisubstrate analog N-phosphonacetyl-L-aspartate at 2.1 Å," Proteins: Struct. Funct. Genet., 37, 729-742.

Macol, C. P., Tsuruta, H., Stec, B., and Kantrowitz, E. R. (2001) "Direct structural evidence for a concerted allosteric transition in Escherichia coli aspartate transcarbamoylase," Nat. Struc. Biol.8, 423-426.

Kelley-Loughnane, N., and Kantrowitz, E. R. (2001) "Binding of AMP to two of the four subunits of pig kidney fructose-1,6-bisphosphatase induces the allosteric transition," PROTEINS, 44, 255-261.

Stieglitz, K. A., Pastra-Landis, S. C., Xia, J., Tsuruta, H., and Kantrowitz, E. R. (2005) "A Single Amino Acid Substitution in the Active Site of Escherichia coli Aspartate Transcarbamoylase Prevents the Allosteric Transition" J. Mol. Biol.  349, 413-423