V-ATPases pump protons into the interior of various subcellular compartments at the expense of ATP. Previous studies have shown that these pumps comprise a membrane-integrated, proton-translocating (V 0 ), and a soluble catalytic (V 1 ) subcomplex connected to one another by a thin stalk region. We present two three-dimensional maps derived from electron microscopic images of the complete V-ATPase complex from the plant Kalanchoë daigremontiana at a resolution of 2.2 nm. In the presence of a non-hydrolyzable ATP analogue, the details of the stalk region between V 0 and V 1 were revealed for the first time in their three-dimensional organization. A central stalk was surrounded by three peripheral stalks of different sizes and shapes. In the absence of the ATP analogue, the tilt of V 0 changed with respect to V 1 , and the stalk region was less clearly defined, perhaps due to increased flexibility and partial detachment of some of the peripheral stalks. These structural changes corresponded to decreased stability of the complex and might be the initial step in a controlled disassembly. V-ATPases1 are found in all eukaryotic cells. They hydrolyze ATP to pump protons into various intracellular compartments (1). In plant tonoplasts the proton motive force generated by the V-ATPase is used for secondary transport processes contributing to osmoregulation, ion and pH homeostasis, nutrient and remnant storage, and plant defense (2).V-ATPases are highly conserved among species, and their gross architecture is similar to that of the well characterized F-ATPases. In the V-ATPases, the soluble V 1 subcomplex is known to carry the catalytic nucleotide-binding sites and to be connected via a thinner stalk region to the membrane-integrated V 0 subcomplex, which contains the proton-translocating machinery. The exact subunit composition and stoichiometry of V-ATPases, however, is still controversial. In yeast, the V 1 subcomplex is probably formed by the subunits (AB) 3 , C-H, and the V 0 subcomplex by the subunits c, cЈ, cЉ, a, and d (for review see Ref.3). Homologues of the cЈ-and cЉ-subunits have not been identified in plants as of yet (4).Among the subunits known to comprise V-ATPases, several share significant sequence homology to subunits of F-ATPases.The catalytic A-subunits of V-ATPase are homologous to the catalytic ␤-subunits in F-ATPase (5) and the B-subunits of V-ATPase to the non-catalytic ␣-subunits in F-ATPase (6). The membrane-integrated V-ATPase c-subunit has probably emerged by gene duplication (7) from a common ancestor of Fand V-ATPases. The G-subunit of V-ATPases has sequence similarity to the hydrophilic part of the membrane-anchored F-ATPase b-subunit (8). Other components of the F-ATPase machinery do not have any homologues in V-ATPase. Furthermore, V-ATPases contain various subunits (C, F, H, a, and d) whose functions and relationships to F-ATPases still need to be elucidated. This divergence might reflect an adaptation to the different physiological requirements of F-and V-ATPases. Unlike F-ATPases, V-AT...

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Substrate mimetics are excellent tools for protease-mediated peptide synthesis that enable the coupling of peptides independently of the primary specificity of the enzyme without undesired cleavages of the newly formed peptide bonds. However, the synthetic utility of this beneficial approach is limited to reactions with nonspecific amino-acid-containing peptides while the coupling of specific ones leads to unwanted cleavages due to the native proteolytic activity of the biocatalyst. This paper reports on the use of site-directed mutagenesis to design trypsin variants with decreased cleavage activity. Starting from the variant D189S, which is known for its low proteolytic potential, Ser189 and Ser190 were exchanged for Ala to further repress the inherent amidase activity of trypsin D189S. The effect of mutations was analysed by model synthesis reactions using specific amino-acid-containing peptides and substrate mimetics as the reactants. Finally, computer-assisted protein-ligand docking studies were performed to get closer insight into the molecular basis of the experimental results.Keywords: peptide synthesis; protein-ligand docking; site-directed mutagenesis; substrate mimetics; trypsinOwing to their reverse hydrolysis activity, proteases can be used as biocatalysts for peptide bond formations, apparently opposite to their original in vivo function [1,2]. The high degree of regio-and stereospecificity of these enzymes allows for protease-mediated peptide couplings free from racemization not requiring large experimental efforts to protect side chains of trifunctional amino acids. Thus, protease-based peptide fragment coupling combined with chemical solution-and in particular solid-phase peptide synthesis represents an attractive route to provide access to larger peptides and proteins [3]. While the general applicability of this semisynthetic strategy could be already demonstrated, the classical enzymatic approach requires very careful planning and optimization of reaction conditions to handle limited enzyme specificities and unwanted proteolytic side reactions. The concept of substrate mimetics is a powerful strategy to overcome this limitation [4±7]. Contrary to common acyl donors, substrate mimetics allow the acylation of the protease by nonspecific acyl residues as the site-specific amino-acid moiety for the enzyme is transferred into the leaving group. Deacylation of the artificial acyl enzyme intermediate by the amino component added results in peptide bond formation regardless of the primary specificity of proteases. Thus, coupling of nonspecific coded and noncoded amino-acid derivatives and even nonamino-acid-derived acyl moieties is achieved. Moreover, as nonspecific amino-acid residues are coupled, the newly formed peptide bond is not subject to secondary hydrolysis. Because of these characteristics, substrate mimetics have proved to be useful for protease-mediated peptide ligations, synthesizing peptide isosteres as well as nonpeptidic carboxylic acid amidesHowever, one limitation of the general appli...