Chemistry Department

Saint Mary's College of California




RESEARCH I am currently investigating the structure function relationship of a Zn-dependent peptidase called Thimet oligopeptidase (TOP). TOP is a soluble enzyme that cleaves small peptides that act as chemical messengers within and between cells1. TOP has been isolated in a numerous cells types, with highest amounts in the brain, testis, and pituitary, and is able to cleave a number of structurally unrelated peptides2. Based on these findings, TOP is believed to regulate several neurological and physiological processes. Furthermore, TOP activity affects the levels of Aβ peptides which are associated with plaque formation in Alzheimer’s disease. Based on the multitude of roles played by TOP, it is relevant to understand the structural mechanism by which it can accommodate its various substrates. A precise understanding of how the enzyme binds to peptides can also aid in the design of potent peptide inhibitors that are needed to follow the physiological activity of the enzyme.

Structural studies have shown that TOP is composed of two domains (see picture above). The substrate binding pocket is formed by a deep channel separating the domains3. The two domains are connected by flexible loops at four regions of the enzyme. The loop structure comprised of residues 599-611 is of particular interest because it is close enough to the active site to interact with even the smallest TOP substrates. This segment includes three glycine residues and should therefore be relatively flexible. Based on the inherent flexibility of this loop and on differences in this region between the crystal structures of TOP and its homolog neurolysin (60% sequence identity), it has been proposed that conformational changes in this segment, and in other loop and coil structures that connect the two domains, lead to changes in subsite specificity necessary to accommodate the numerous peptide substrates hydrolyzed by TOP3.

We are currently trying to determine how conformational changes in the structure of TOP lead to changes in substrate specificity. Site-directed mutagenesis has been used to change specific glycine residues in the TOP loop( 599-611) to alanine residues, and activity assays to monitor the changes in activity of the enzyme towards several physiologically relevant peptides. Alanine is the smallest protein residue next to glycine, yet does not possess the inherent rotational flexibility of glycine. Therefore, a glycine to alanine mutation can trap the loop in a specific conformation. Three glycine residues 599, 603, and 604 have been individually changed allowing study of various conformations of the loop and the affect on TOP activity. This is analogous to changing a lock and then testing it for different shaped keys. The activity of TOP is monitored by use of quenched fluorescence substrates. These are peptides that contain an N-terminal fluorescent tag and a C-terminal quencher. As TOP cleaves the peptide, the fluorescent probe and the quench er are separated and drift apart in sol ution, there is a large increase in the fluorescence intensity of the probe. In this fashion, one can obtain rate constants for the cleavage of the peptide under various conditions.

Our previous results have shown that specific conformational changes in TOP, induced by the chemical denaturant urea, lead to changes in substrate specificity for two quenched fluorescence substrates. Furthermore, our initial results with two of the glycine to alanine mutants have revealed that the two mutants have differential affects on the activity towards one of these substrates. This information can shed insight onto the structural basis by which TOP can play a role in many physiological processes such as blood pressure control, reproduction, and the immune response.

1. Shrimpton, C. N.; Smith, A. I.; Lew, R. A., Soluble metalloendopeptidases and neuroendocrine signaling. Endocr. Rev. 2002, 23, (5), 647-64.

2. Chu, T. G.; Orlowski, M., Solub le metalloendopeptidase from rat brain: action on enkephalin-containing peptides and other bioactive peptides. Endocrinology 1985, 116, (4), 1418-25.

3. Ray, K.; Hines, C. S.; Coll-Rodriguez, J.; Rodgers, D. W., Crystal structure of human thimet oligopeptidase provides insight into substrate recognition, regulation, and localization. J. Biol. Chem. 2004, 279, (19), 20480-9.