The metalloproteolytic activity of the anthrax lethal factor is substrate-inhibited

The anthrax lethal factor (LF) is a Zn 2+ -endopeptidase specific for mitogen-activated protein kinase kinases (MAPKKs), which are cleaved within their N -termini. Here, the proteolytic activity of LF has been investigated using novel chromogenic MAPKK-derived peptide substrates, which allowed us to determine the kinetic parameters of the reaction. LF displayed maximal proteolytic activity at the pH and temperature values of the cell cytosol, which is its site of action. LF undergoes substrate inhibition, in keeping with the non-productive binding geometry of the MAPPK-2 N terminus to LF.


Introduction
Toxigenic strains of Bacillus anthracis secrete three proteins: the protective antigen (PA, 87.2 kDa), the edema factor (EF, 88.8 kDa), and the lethal factor (LF, 90.2) [1][2][3]. PA derives its name from the fact that immunization with it confers a protective immunity to vaccinated animals [4][5]. PA binds to a rather ubiquitous plasma membrane type I protein encoded by the tumor endothelial marker gene 8 [6][7] and to human capillary morphogenesis protein 2 [8]. PA is then proteolytically processed on the cell surface by furin and membrane furin-like peptidases, to a 63 kDa form (PA 63 ) which oligomerizes and binds LF or EF [3].
The lethal toxin (PA + LF) and the edema toxin (PA + EF) are then endocytosed by a lipid raft-mediated clathrin-dependent process [9]. Then, PA 63 undergoes an acidic triggered conformational rearrangement [10] that mediates the transfer of EF or LF from the lumen of a late endocytic compartment to the cytoplasm [11].
EF is a Ca 2+ -and calmodulin-dependent adenylate cyclase that increases cytosolic cAMP, altering water homeostasis and the elaborate balance of intracellular signaling pathways. EF impairs neutrophil function(s) and it is believed to be responsible for the edema found in cutaneous anthrax [12].
LF displays metalloproteolytic activity directed toward the N-terminus of mitogenactivated protein kinase kinases (MAPKKs) [13][14][15][16]. The active site zinc ion is coordinated tetrahedrally to the domain 4 of LF (residues 551-777) by a water molecule and three side chains (i.e. H686, H690, and E735), in a structural arrangement shared by metalloproteases of the thermolysin family [17]. LF has a deep and long (~40 Å long) groove contiguous to the active site center which binds peptide substrates and peptide inhibitors [17][18]. The groove has an overall negative electrostatic potential containing clusters of E/D as well as Q/N residues. The determination of the sites of proteolysis of various MAPKK isoforms led to the identification of a consensus motif for the cleavage site: positively charged residues are located at positions P 7 to P 4 and hydrophobic residues at P 2 and P 1 '; in addition, an aliphatic hydrophobic residue is present at position P 1 (Fig. 1) 1 [15].
The lethal toxin metalloproteolytic activity is cytotoxic to macrophages [19] and there is evidence that it plays a major role in the development of systemic anthrax, a rapid and often fatal disease of several vertebrates including humans [20]. It is worth noting that LF active site mutants are non toxic [21] and membrane permeable metalloprotease inhibitors prevent the LF cytotoxic activity on cultured macrophages [18].
Here, we present a detailed analysis of the LF catalyzed hydrolysis of chromogenic substrates designed on the basis of the consensus motif of MAPKK N-termini and of the inhibition caused by the substrates themselves. These findings are relevant to the design of novel inhibitors of LF and for the understanding of the macrophage toxicity of LF.

LF and peptide chromogenic substrate synthesis
Recombinant LF was prepared as previously reported and the LF concentration was determined by absorbance at 280 nm (E 0.1% 1 cm = 0.798) [15]. Small aliquots were frozen in liquid nitrogen and stored at -80°C. Peptides from the Leu P 2 residue to the Nterminal amino acid (
The LF catalyzed hydrolysis of peptide chromogenic substrates was analyzed in the framework of the minimum mechanism for total substrate inhibition (Scheme 1) [26][27]:  [26,27]:

Results and Discussion
On the basis of the knowledge of the LF:MAPKK recognition properties [15], the LF peptide chromogenic substrates listed in Table 1  As shown in Figure 2, LF undergoes total substrate inhibition, indicating that the substrate may bind to the enzyme with different geometries, the substrate bound in the nonproductive binding mode(s) impairs LF action. Total substrate inhibition is in keeping with the observation that the LF:MAPKK-2 substrate adduct is the first example of a protease in complex with its uncleaved substrate [17]. It is worth noting that although the closest mainchain approach to the Zn 2+ ion is the scissile bond following MAPKK-2 P10 (i.e. the P 1 -P 1 ' bond), it is about 6 Å from the zinc-bound water. Modelling studies have shown that a rotation about a main-chain dihedral angle at K6 (i.e. P 5 ) would allow P10 (i.e. P 1 ) to swing down into the S 1 specificity subsite to generate the productive cleavage complex. The substrate productive conformation requires an abrupt 90° turn in the peptide chain (favoured by the P10 residue) at the P 1 position (Fig. 1) [17].
The analysis of the data shown in Figure 2, according to Eqn 1, provided the values of K m , k cat , and k cat /K m for the LF catalyzed hydrolysis of peptide chromogenic substrates, as well as of K i for enzyme inhibition by the pNA-and AMC-derivatized substrates ( Table 1). The turnover number of LF shows a limited variation (from 2 to 6 molecules of substrate hydrolysed per second by one molecule of LF) upon changing the length of peptide substrates and the number of R residues, whose addition at the N-terminus renders peptides permeable to the plasma membrane of cells [18]. This figure can be compared with that of the prototype metalloproteinase, thermolysin, whose turnover number is comprised in the range 6 to 16 substrate molecules hydrolysed per enzyme molecule [28]. can be taken as an additional evidence of the importance of electrostatic interactions in LFsubstrate recognition. This is in agreement with the decrease of the initial velocity (i.e. v i ) for the LF catalyzed hydrolysis of the peptide chromogenic substrate AcMLARRRPVLP-pNA on increasing the ionic strength (i.e. the NaCl concentration) (Fig. 3). It should be noted that the negatively charged residues E336, E334, D387, and D394 located in the elongated cleftshaped recognition site of LF (Fig. 1) are in such a position within the active site as to suggest that they interact directly with the substrate N-terminal positively charged R residues thus playing a major role in the binding and the positioning of the substrate [17].
As shown in Table 1, values of k cat for the LF catalyzed hydrolysis of peptide chromogenic substrates are essentially substrate-independent (Table 1). This may reflect a common rate limiting step in catalysis. As shown in Figure 3, the maximum initial velocity (i.e. maximum v i ) for the LF catalyzed hydrolysis of AcMLARRRPVLP-pNA occurs at pH 7.4 and 37°C, corresponding to pH and temperature values of the cytosol of mammalian cells, which is the site of action of LF.
It should be noted that values of k cat for the LF catalyzed hydrolysis of the peptide substrates conjugated with pNA or with AMC, which differ considerably in size, are almost identical (Table 1). This is in agreement with the previous findings [15,17] that the main determinants of the substrate binding into the active site cleft of LF are located on the Nterminus side. The AMC-derivatized substrate AcRRRRVLR-AMC offers a much higher sensitivity than the pNA-derivatized substrates allowing one to follow the proteolytic activity of minute amounts of LF (about one hundred fold less enzyme is needed). The hydrolysis of both types of substrates can be monitored with very simple apparatuses and in a wide range of experimental conditions, including high throughput assays.

Conclusions
This is the first detailed study of the kinetic parameters of the metalloproteolytic activity of LF in vitro obtained with ad hoc designed chromogenic substrates whose hydrolysis can be monitored with very simple and widely available techniques. It describes the substrate inhibition of LF hydrolytic activity. The subtle modulation of LF activity by total substrate inhibition is in keeping with the non-productive binding mode of MAPKK-2 Nterminus to LF [17] and could be relevant for the enzyme action in vivo. In fact, LF cleaves several isoforms of MAPKK present within the cytosol of cells. Given the different structural properties of these cellular substrates and their different sub-cellular locations [29,30], it is very likely that they are cleaved with different velocities, and that there is a non-uniform LFinduced cleavage of the MAPKK isoforms. Such heterogeneity of cleavage of MAPKK isoforms could additionally vary among cells giving rise to different cellular outcomes of the intoxication with LF. Indeed, the effect of LF varies with cell types and conditions. Upon exposure to PA+LF, macrophages lyse in culture [19,31], but die from apoptosis if they are primed with lipopolysaccharides or tumor necrosis factor-α [32,33]. On the other hand, other cells are resistant though their MAPKK-3 is cleaved by LF [15,34,35]. A quantitative analysis of the differential LF cleavage of MAPKK isoforms within cells is not a simple goal to achieve, but it appears to be essential for the molecular understanding of the in vivo action of LF.

Footnotes
Footnote to page 4: Peptide substrates k cat (s -1 ) K m (µM) k cat /K m (sec -1 µΜ −1 ) K i (µΜ) 8 P 7 P 6 P 5 P 4 P 3 P 2 P 1 P 1 '     Substrate site P 8 P 7 P 6 P 5 P 4 P 3 P 2 P 1 P 1 ' P 2 ' P 3 ' P 4 ' P 5 ' P 6 ' P 7 ' P 8 ' MAPKK-1 (P 8 -I 9 ) M P K K K P T P I Q L N P A P D MAPKK-2 (P 10 -A 11 ) A R R K P V L P A L T I N P T I MAPKK-3b (R 26 -I 27 ) S K R K K D L R I S C M S K P P MAPKK-6b (K 14 -I 15 ) K K R N P G L K I P K E A F E Q MAPKK-4 (K 45 -L 46 ) Q G K R K A L K L N F A N P P F MAPKK-4 (R 58 -F 59 ) P P F K S T A R F T L N P N P T MAPKK-7 (Q 44 -L 45 ) Q R P R P T L Q L P L A N D G G MAPKK-7 (G 76 -L 77 ) A R P R H M L G L P S T L F T P