On the reaction mechanism of legumain at atomistic details

The subject of this study is the enzyme legumain, which catalyzes the cleavage and/or degradation of proteins under acidic conditions in a two-step procedure. Since tumors are also proteins, and many of them are having acidic bio-environment, the decomposition of tumors may allow such activity. Therefore this enzyme has been utilized for experimental pro-drug activation ensuring tumor- targeted delivery of chemotherapeutic drugs. Since experimental work cannot provide in depth information about the catalytic reaction, the exact mechanism of action has not been clarified yet. Here, we propose to study the reaction mechanism of legumain at atomistic details using high level computational quantum chemistry methods. Starting from the X-ray structure of the enzyme-inhibitor complex (legumain-cystatin) both the acylation and deacylation process will be investigated step-by-step by applying harmonic restraints (springs) to cleave or to generate the bond of interest. This technique allows us to drive the system towards the desired intermediate and product states. Afterwards the transition state structures and the reaction free energy will be calculated to determine the rate limiting step and the reaction barrier of the catalytic cycle. In addition we intend to explain the findings of site directed mutagenesis studies and the pH dependency. We propose to consider several alternative reaction paths and compare the calculated reaction energies with the experimentally measured value Due to the fundamental biological activity of legumain in cancer therapy the understanding of the full catalytic mechanism is indispensable in order to be able to develop effective pro-drug activated chemotherapeutic agents.

Depending on the pH of the environment he cysteine protease legumain catalyzes the cleavage (pH 4.0) and/or ligation (pH 6.0) of proteins with preference for substrate hydrolysis C-terminally to asparagine and ev. after aspartic acid. In this study, using high level quantum chemical calculations, we could clarify the pH-dependent dual activity of human legumain.Our simulations were based on the crystal structure of human legumain in complex with human cystatin E. Our calculations have shown that the protonation states of the catalytic cysteine (Cys189) and histidine (His148) are crucial for the reaction mechanism to proceed either towards cleavage or ligation. The most important milestones concerning the reaction mechanism of the cleavage and ligation are:The cleavage starts with a protonated Cys189 and neutral His148The proton from Cys189 transfers to the nitrogen of the scissile peptide bondThere is no papain-like classical intermediate was found since the first intermediate is already the acyl-enzymeWater attack on the thioester results in a diol (second stable intermediate), where the nucleophile is OH The role of the catalytic His148 is to serves as base to the diol during product formationThe ligation step can proceed as a one-step procedure without cysteine participation. This surprising fact has also been proven experimentally both by blocking Cys189 using MMTS and by applying the point mutation C189M. Only legumain with deprotonated Cys189 can act as a ligase, which is consistent with the experimental pH profile of the reaction. The exact reverse reaction of the proteolysis is not possible, since in the present of the catalytic water the sulphur of the thioester is not strong enough as base to remove the proton from the N-terminus. However, transpeptidation via thioester is possible if there is no catalytic water available at the active site.Moreover, this is first time that the complete reaction pathways of both enzymatic activities are presented and is in excellent agreement with experimental data. Due to the fundamental biological activity of legumain in cancer therapy the understanding of the full catalytic mechanism is indispensable in order to be able to develop effective pro-drug activated chemotherapeutic agents.