Molecular Dynamics Simulations of the Catalytic Pathway of a Cysteine Protease:  A Combined QM/MM Study of Human Cathepsin K

Abstract

Molecular dynamics simulations using a combined QM/MM potential have been performed to study the catalytic mechanism of human cathepsin K, a member of the papain family of cysteine proteases. We have determined the two-dimensional free energy surfaces of both acylation and deacylation steps to characterize the reaction mechanism. These free energy profiles show that the acylation step is rate limiting with a barrier height of 19.8 kcal/mol in human cathepsin K and of 29.3 kcal/mol in aqueous solution. The free energy of activation for deacylation step is 16.7 kcal/mol in cathepsin K and 17.8 kcal/mol in aqueous solution. The reduction of free energy barrier is achieved by stabilization of the oxyanion in the transition state. Interestingly, although the "oxyanion hole" has been formed in the Michaelis complex, the amide units do not donate hydrogen bonds directly to the carbonyl oxygen of the substrate, but they stabilize the thiolate anion nucleophile. Hydrogen bonding interactions are induced as the substrate amide group approaches the nucleophile, moving more than 2 angstroms and placing the oxy-anion in contact with Gln19 and the backbone amide of Cys25. The hydrolysis of peptide substrate shares a common mechanism both for the catalyzed reaction in human cathepsin K and the uncatalyzed reaction in water. Overall, the nucleophilic attack by Cys25 thiolate and the proton transfer reaction from His162 to the amide nitrogen are highly coupled, whereas a tetrahedral intermediate is formed along the nucleophilic reaction pathway.

Structure of the Mature Streptococcal Cysteine Protease Exotoxin mSpeB in Its Active Dimeric Form

“…Gln is part of the oxyanion hole, Cys is the catalytic nucleophile and His stabilizes the thiolate ion in the ground state as well as acts as an acid catalyst during acylation and as a base catalyst during deacylation. 12 The Asp or Asn residue acts to position the His via accepting a hydrogen bond from the imidazolium nitrogen located distal to the nucleophile. This Asp/Asn residue is not present in all families; in some enzymes, including SpeB, this functionality is fulfilled by a backbone oxo group.…”

Effect of altering local protein fluctuations using artificial intelligence

“…As a model protein we selected the cysteine protease cathepsin K, a notable curative agent for osteoporosis and rheumatoid arthritis. 12 The effects of locally regulating cathepsin K fluctuations were examined in 15 model ligands. The ligands were dipeptides selected from five residues, and their molecular weights approximately that of E64 (the typical inhibitor of cysteine protease).…”

FEARCF a multidimensional free energy method for investigating conformational landscapes and chemical reaction mechanisms

“…Recent modifications allow the calculation of a variety of reaction coordinate types besides the distances I have used here in this application [17]. Although an early version of the procedure [15] has been shown to be useful in applications to enzymatic reactions involving complex reaction mechanisms that require the distinction between concerted and step-wise processes [49][50][51].…”