DOI: 10.1002/chem.201301310 Understanding the Autocatalytic Process of Pro-kumamolisin Activation from Molecular Dynamics and Quantum Mechanical/Molecular Mechanical (QM/MM) Free-Energy Simulations Jianzhuang Yao, [a] Alexander Wlodawer, [b] and Hong Guo* [a] Proteolytic enzymes are synthesized as inactive precursors or zymogens to promote folding, prevent unwanted protein degradation, and provide a mechanism for regulating pro- tein function through proteolytic activation. Understanding the ways by which Nature prevents unwanted activation of proteases and mechanisms for conversion of zymogens to active enzymes is of considerable interest. [1] However, a de- tailed understanding of the activation processes and energet- ics involved is still lacking. Herein, we applied molecular dy- namics (MD) and quantum mechanical/molecular mechani- cal (QM/MM) free-energy simulations to study the process and energetics of the conversion of the kumamolisin zymo- gen to the active enzyme. It has been shown that the proto- nation of Asp164 would trigger conformational changes and generate the functional active site for autocatalysis. The mechanism of acylation for autocatalytic cleavage of prodo- main is also derived from the two-dimensional QM/MM free-energy simulations. The results seem to indicate that one of the reasons for sedolisins to use an aspartate as a cat- alyst (e.g., Asp164 in kumamolisin) instead of asparagine (the oxyanion-hole residue in classical serine peptidase) might be due in part to the requirement for the creation of a built-in switch that delays the self-activation until secre- tion into acidic medium. Kumamolisin belongs to a recently characterized family of serine-carboxyl peptidases (sedolisins), [2, 3] which are present in a wide variety of organisms and are most active at low pH. The members of the family also include tripeptidyl-pep- tidase 1 (TPP1) for which the loss of the activity as a result of mutations in the TPP1 gene is believed to be the cause of a fatal neurodegenerative disease. [4] Similar to other proteo- lytic enzymes, sedolisins are synthesized as inactive precur- sors to prevent unwanted activation and proteolysis. The in- active precursors for sedolisins include propeptides of ap- proximately 200 amino acids in length, which may play a role in the folding of the proteins, as well as protecting them from being prematurely activated. The activation cleavage of the prodomains occurs after the release of zymogens into the acidic environment, leading to the production of the active enzymes. [3b] The crystal structures have been determined for the inac- tive Ser278Ala pro-kumamolisin mutant, [5] as well as for the active kumamolisin. [6] A superposition of the structure of the Ser278Ala pro-kumamolisin mutant with that of kuma- molisin showed that the catalytic domain of the mutant ex- hibited a virtually identical structure compared to the active enzyme. [5] The pro-kumamolisin structure has therefore proved that the catalytic domain has basically adopted a mature-like conformation already in the zymogen form. One of the key structural features of the proenzyme is the presence of a salt bridge between the P 3 -Arg169p linker res- idue (herein, the suffix “p” designates the residues in the pro-domain) and Asp164 from the catalytic domain. [5] The existence of this salt bridge seems to prevent the formation of the functional configuration for Asp164. [7] An interesting question is whether the protonation of Asp164 and subse- quently breaking of this salt bridge during secretion into an acidic medium would trigger conformational changes and lead to generation of well-positioned general acid/base cata- lyst (Asp164) and creation of a functional active site in the preparation of the self-activation. [5] The X-ray structure of the Ser278Ala pro-kumamolisin mutant was used to generate the model for the wild-type pro-kumamolisin through a manual change of Ala278 to Ser278. Figure 1 A shows that the catalytic Ser residue, along with Glu78 and Asp82, are located at the correct posi- tion and would attack the carbonyl carbon of P 1 -His171p during the autocatalytic cleavage. This agrees with the earli- er suggestion that the catalytic triad in the pro-kumamolisin structure has already adopted a mature-like conformation. [5] In contrast, Asp164 is located at a different position com- pared to that observed in the active enzyme [6] and forms a salt bridge with the P 3 -Arg169p (Figure 1 A). Asp164 is therefore unable to participate in the stabilization of the TI during the autocatalytic cleavage of the peptide bond be- tween P 1 -His171p and P 1 ’-Phe172p. [a] J. Yao, Prof. H.Guo Department of Biochemistry and Cellular and Molecular Biology University of Tennessee, Knoxville, TN 37996 (USA) Fax: (+ 1) 865-974-6306 E-mail : hguo1@utk.edu [b] Dr. A. Wlodawer Protein Structure Section Macromolecular Crystallography Laboratory National Cancer Institute, Frederick, MD 21702 (USA) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201301310. Chem. Eur. J. 2013, 19, 10849 – 10852  2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 10849 COMMUNICATION