The Catalytic Mechanism of an Aspartic Proteinase Explored with Neutron and X-ray Diffraction

Abstract

Aspartic proteinases are a class of enzymes widely distributed among fungi, plants, vertebrates, and viruses. They are involved in numerous disease conditions, including hypertension, amyloid disease, malaria, and AIDS. 1 In HIV, the proteinase is essential for the maturation of the virus particle, and inhibitors have a proven therapeutic effect in the treatment of AIDS. There have also been significant recent advances in the development of orally active renin inhibitors which are in clinical trials. 2 Thus inhibitors to this class of enzyme with improved characteristics are therefore much sought after as potential therapeutic agents. The aspartic proteinase class of enzyme comprises two structurally similar domains, each contributing an aspartic acid residue to form a catalytic dyad that cleaves the substrate peptide bond. The two catalytic aspartate carboxyls are held coplanar by a network of hydrogen bonds involving the surrounding main chain and the conserved amino acid side-chain groups. Most aspartic proteinases are inhibited by pepstatin, a microbial peptide that contains the unusual amino acid statine, and a number of crystal structures of complexes containing this naturally occurring analogue have been reported. 3,4 Endothiapepsin, a member of the aspartic proteinase family, is composed of 330 amino acid residues, with roughly 170 residues in each domain. Endothiapepsin is derived from the fungus Endothia parasitica, and numerous structures of this enzyme bound to a range of renin inhibitors have been analyzed with the aim of developing improved compounds by structure-based design. 5 Crystallographic studies of all native aspartic proteinases have revealed a water molecule hydrogen bonded to the carboxyl groups of both aspartate residues. This water molecule is within hydrogen-bonding distance of all four carboxyl O atoms and has been implicated in catalysis. It has been suggested 6 that this water is partly displaced upon substrate binding and is polarized by one of the catalytic aspartate residues. It then initiates enzymatic catalysis by attacking the scissile bond carbonyl group of the substrate. The transition state of aspartic proteinase catalysis has been studied most extensively using enzyme-inhibitor complexes which possess a range of different transition state mimics. Most inhibitors mimic one or both hydroxyls in the putative transition state. The statine-based inhibitors contain one hydroxyl that occupies the same position as the water E-mail: coatesl@ornl.gov. † Oak Ridge National Laboratory. ‡ University of Toledo. § Los Alamos National Laboratory. ¶ University College London.Supporting Information Available: Data collection and refinement statistics for all three structures discussed within this paper, the atomic resolution, the room temperature X-ray, and room temperature neutron. This material is available free of charge via the Internet at http://pubs.acs.org. Figure 1. NIH Public AccessResults from this study suggest that the transition state is stabilized by a negative charge on Asp...

Transition states of native and drug-resistant HIV-1 protease are the same

“…However, little change occurs between 10, 13, and 11 (in order of decreasing r ðN-HÞ ), with r ðC-NÞ changing only slightly from 1.51, 1.52, to 1.53 Å, respectively. Hydrogen bonding to both oxygens of the gem-diol occurs solely with Asp-125 and is maintained throughout the reaction, consistent with previous dynamic, crystallographic, and neutron diffraction studies (15).…”

“…We have measured primary 14 C and 15 N KIEs and secondary 3 H and 18 O KIEs for native and multidrug-resistant HIV-1 protease (I84V). We observed 14 C KIEs ( 14 V∕K) of 1.029 AE 0.003 and 1.025 AE 0.005, 15 N KIEs ( 15 V∕K) of 0.987 AE 0.004 and 0.989 AE 0.003, 18 O KIEs ( 18 V∕K) of 0.999 AE 0.003 and 0.993 AE 0.003, and 3 H KIEs ( 3 V∕K) KIEs of 0.968 AE 0.001 and 0.976 AE 0.001 for the native and I84V enzyme, respectively. The chemical reaction involves nucleophilic water attack at the carbonyl carbon, proton transfer to the amide nitrogen leaving group, and C-N bond cleavage.…”

“…The cycle initiates with water activation by hydrogen-bonding interactions with the active site aspartate residues (1) (6, 7). Bell-shaped pH-rate profiles (8)(9)(10)(11)(12) have revealed an acidic pH optimum, and structural studies (13,14) suggest that the reactant-bound enzyme contains a single protonated Asp (with a di-Asp, net charge of −1) facilitating a general acid-base mechanism, which is common among many Asp proteases (7,15). Nucleophilic attack by the water generates a reversible gem-diol intermediate, (3) which has been observed structurally (16)(17)(18) and identified experimentally (19,20).…”

Fragment Linking and Optimization of Inhibitors of the Aspartic Protease Endothiapepsin: Fragment‐Based Drug Design Facilitated by Dynamic Combinatorial Chemistry

“…This class of enzymes play a causative role in important diseases such as malaria, Alzheimer's disease, hypertension, and AIDS 23. Owing to its high similarity with these drug targets, endothiapepsin has been used as a model enzyme for mechanistic studies24, 25, 26 and for the discovery of inhibitors of renin27 and β‐secretase 28. Endothiapepsin is a robust enzyme, which remains active for more than 20 days at room temperature, is readily available in large quantities, and crystallizes easily, thus making it a useful representative for aspartic proteases 18.…”

Direct observation of hydrogen atom dynamics and interactions by ultrahigh resolution neutron protein crystallography

“…The scattering properties of H and D allow for the accurate placement of these atoms in small molecule and macromolecular structures, in contrast to X-rays, where the single electron and proximity to heavier atoms make H atoms difficult to resolve. Moreover, the scattering characteristics of D have useful applications in neutron macromolecular crystallography, such as determining protonation states of active site residues in enzymes and establishing the identities and orientation of solvent molecules (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Neutron diffraction has also been useful for determining orientations of methyl rotors and probing the energy barrier to rotation about a chemical bond (16,17).…”