Crystallographic and Biochemical Investigations of Kumamolisin-As, a Serine-Carboxyl Peptidase with Collagenase Activity

Abstract

Kumamolisin-As (previously called ScpA) is the first known example of a collagenase from the sedolisin family (MEROPS S53). This enzyme is active at low pH and in elevated temperatures. In this study that used x-ray crystallographic and biochemical methods, we investigated the structural basis of the preference of this enzyme for collagen and the importance of a glutamate residue in the unique catalytic triad (Ser 278 -Glu 78 -Asp 82 ) for enzymatic activity. Crystal structures of the uninhibited enzyme and its complex with a covalently bound inhibitor, N-acetyl-isoleucyl-prolyl-phenylalaninal, showed the occurrence of a narrow S2 pocket and a groove that encompasses the active site and is rich in negative charges. Limited endoproteolysis studies of bovine type-I collagen as well as kinetic studies using peptide libraries randomized at P1 and P1, showed very strong preference for arginine at the P1 position, which correlated very well with the presence of a negatively charged residue in the S1 pocket of the enzyme. All of these features, together with those predicted through comparisons with fiddler crab collagenase, a serine peptidase, rationalize the enzyme's preference for collagen. A comparison of the Arrhenius plots of the activities of kumamolisin-As with either collagen or peptides as substrates suggests that collagen should be relaxed before proteolysis can occur. The E78H mutant, in which the catalytic triad was engineered to resemble that of subtilisin, showed only 0.01% activity of the wild-type enzyme, and its structure revealed that Ser 278 , His 78 , and Asp 82 do not interact with each other; thus, the canonical catalytic triad is disrupted.A novel peptidase, initially named ScpA (1) and now called kumamolisin-As (2), was recently identified by us in the culture filtrate of a thermoacidophilic soil bacterium Alicyclobacillus sendaiensis strain NTAP-1 (1). Specificity analyses using macromolecular substrates including globular and other fibrillar proteins showed that kumamolisin-As is highly specific for collagen (3, 4) and thus could be considered as a collagenase, although with some unusual properties. Most noticeably, this enzyme exhibits the maximum activity at acidic pH ϳ4.0. This is in striking contrast to all known collagenases, which are either zinc-dependent metallopeptidases (5) or chymotrypsinlike serine proteinases (6, 7), with an optimum pH for activity at neutral to alkaline regions.A primary structure analysis of this novel "acid collagenase" revealed that it is a member of the sedolisin family, a recently established class of serine peptidases with a unique catalytic triad, Ser-Glu-Asp, in place of the Ser-His-Asp triad of classical serine peptidases (2, 4). Moreover, the enzyme was found to be very similar in its primary structure to kumamolisin, a well characterized member of the family (8 -10), exhibiting 92.7% identity with its mature form. This high level of identity led to the change of the name from the initially used ScpA (1) to kumamolisin-As (2). Kumamolisin-As was the fir...

The QM/MM Molecular Dynamics and Free Energy Simulations of the Acylation Reaction Catalyzed by the Serine-Carboxyl Peptidase Kumamolisin-As

“…For instance, it was found that the k cat value for a substrate containing the -ProArg*Gly sequence is ∼65-fold higher than for a substrate containing the -Pro-Gly*Gly sequence and the K M is ∼6.5-fold higher, leading to 10-fold increase of k cat /K M (3). The higher specificity for the substrates with a positively charged residue at P 1 was also confirmed from specificity profile analysis using a peptide library (9). It was suggested in our earlier computational study (16) that the dynamics involving His at P 1 triggered by the bond-breaking and -making events may play an important role in the stabilization of the tetrahedral intermediate and therefore contribute to the relatively high specificity for the substrates with His at P 1 .…”

“…The substrate used in the present study, GPH*FF, contains the key residues of the internally quenched fluorogenic (IQF) substrate [NMA-MGPH*FFPK-(DNP)DRDR] used in the earlier experimental study (9,10), although it is considerably smaller. One of the key questions for performing the simulations on kumamolisin-As is how to obtain the coordinates for the substrate (tetrahedral intermediate) in the enzyme complex.…”

“…The average active site structure for the kumamolisin-Assubstrate (GPH*FF) complex obtained from the QM/MM MD simulations is given in Figure 1A. Ser278 is the nucleophile that attacks the carbonyl carbon atom (C) of the substrate, while Glu78 and Asp164 act as the general base and acid catalysts, respectively (4)(5)(6)(7)(8)(9)(10)(15)(16)(17). Two additional residues, Glu32 and Trp129, interact with Asp82 of the catalytic triad through hydrogen-bonding networks.…”

Molecular dynamics suggest multifunctionality of an adenine imino group in acid-base catalysis of the hairpin ribozyme

“…Such a mechanism is analogous to the role of, for example, an active site Glu in the first step of the reaction carried out by serine-carboxyl peptidases ( Fig. 8C; Wlodawer et al 2004;Xu et al 2007). Here, the A-1(29OH) to A38(N1) hydrogen bond would be essential to the mechanism; following transfer of the proton from A-1(29OH) to A38(N1) the protonated A38H+ again promotes formation of the active geometry.…”

“…In both mechanistic proposals G8, A10, A38, and water 5 stabilize the negative charge accumulated in the transition state. (C) Mechanism proposed for the protein-based serine-carboxyl peptidase kumamolisin-As (Wlodawer et al 2004;Xu et al 2007), analogous to the mechanism in panel B. A glutamate side chain with acidic solution pK a (4.3) acts as both general base and acid by shuttling a proton directly from the serine nucleophile with solution pK a of (z13) to the leaving group during formation of the acylated enzyme.…”