Blood-feeding pathogens digest hemoglobin (Hb) as a source of nutrition, but little is known about this process in multicellular parasites. The intestinal brush border membrane of the canine hookworm, Ancylostoma caninum, contains aspartic proteases (APR-1), cysteine proteases (CP-2), and metalloproteases (MEP-1), the first of which is known to digest Hb. We now show that Hb is degraded by a multi-enzyme, synergistic cascade of proteolysis. Recombinant APR-1 and CP-2, but not MEP-1, digested native Hb and denatured globin. MEP-1, however, did cleave globin fragments that had undergone prior digestion by APR-1 and CP-2. Proteolytic cleavage sites within the Hb ␣ and ␤ chains were determined for the three enzymes, identifying a total of 131 cleavage sites. By scanning synthetic combinatorial peptide libraries with each enzyme, we compared the preferred residues cleaved in the libraries with the known cleavage sites within Hb. The semi-ordered pathway of Hb digestion described here is surprisingly similar to that used by Plasmodium to digest Hb and provides a potential mechanism by which these hemoglobinases are efficacious vaccines in animal models of hookworm infection.

CcdA, the antidote protein of the ccd post-segregational killing system carried by the F plasmid, was degraded in vitro by purified Lon protease from Escherichia coli. CcdA had a low affinity for Lon (K m 200 M), and the peptide bond turnover number was ϳ10 min ؊1 . CcdA formed tight complexes with purified CcdB, the killer protein encoded in the ccd operon, and fluorescence and hydrodynamic measurements suggested that interaction with CcdB converted CcdA to a more compact conformation. CcdB prevented CcdA degradation by Lon and blocked the ability of CcdA to activate the ATPase activity of Lon, suggesting that Lon may recognize bonding domains of proteins exposed when their partners are absent. Degradation of CcdA required ATP hydrolysis; however, CcdA41, consisting of the carboxylterminal 41 amino acids of CcdA and lacking the ␣-helical secondary structure present in CcdA, was degraded without ATP hydrolysis. Lon cleaved CcdA primarily between aliphatic and hydrophilic residues, and CcdA41 was cleaved at the same peptide bonds, indicating that ATP hydrolysis does not affect cleavage specificity. CcdA lost ␣-helical structure at elevated temperatures (T m ϳ50°C), and its degradation became independent of ATP hydrolysis at this temperature. ATP hydrolysis may be needed to disrupt interactions that stabilize the secondary structure of proteins allowing the disordered protein greater access to the proteolytic active sites.Viability of bacterial cells harboring unit copy number plasmids is potentially compromised by the presence of plasmidencoded gene products that are toxic to the cell. Cells carrying such plasmids survive because the plasmids encode, usually in the same operon with the toxin, a second gene product that acts as an antidote (reviewed in Refs. 1, 2). The antidote molecule is unstable and has a shorter half-life than the toxin; therefore, long term survival of the cells requires the continuous production of the antidote. In cells that lose the plasmid, antidote concentrations decrease faster than those of the toxin, resulting in killing of the plasmid-cured cells. F plasmid contains three operons that function independently and with varying degrees of effectiveness as post-segregational killing systems (3-5). One such operon, ccd, plays a relatively minor role in post-segregational killing with intact F plasmid but, when present on a mini-F plasmid or when cloned on a plasmid with a heterologous replicon, results in killing of Ն90% of plasmidfree segregants (3, 6, 7). ccd encodes CcdB, an 11-kDa protein that inhibits DNA gyrase (8 -10), and CcdA, a 9-kDa protein that blocks the action of CcdB (6, 11).CcdA is degraded in wild-type cells with a t1 ⁄2 ϳ30 min in the absence of CcdB and a t1 ⁄2 ϳ60 min in the presence of CcdB (12). Because CcdA is expressed in higher amounts than CcdB, it remains in excess of CcdB and neutralizes CcdB activity as long as the plasmid bearing ccd is maintained. Loss of the mini-F plasmid results in a decrease of CcdA over several generations leading to CcdB-mediated cell d...

A remote labeling method has been developed to determine 18 O kinetic isotope effects (KIEs) in Ras-catalyzed GTP hydrolysis. Substrate mixtures consist of 13 C-depleted GTP and [ 18 O, 13 C]GTP that contains 18 O at phosphoryl positions of mechanistic interest and 13 C at all carbon positions of the guanosine moiety. Isotope ratios of the nonvolatile substrates and products are measured by using a chemical reaction interface͞isotope ratio mass spectrometer. The isotope effects are 1.0012 (0.0026) in the ␥ nonbridge oxygens, 1.0194 (0.0025) in the leaving group oxygens (the ␤-␥ oxygen and the two ␤ nonbridge oxygens), and 1.0105 (0.0016) in the two ␤ nonbridge oxygens. The KIE in the ␤-␥ bridge oxygen was computed to be 1.0116 or 1.0088 by two different methods. The significant KIE in the leaving group reveals that chemistry is largely rate-limiting whereas the KIEs in the ␥ nonbridge oxygens and the leaving group indicate a loose transition state that approaches a metaphosphate. The KIE in the two ␤ nonbridge oxygens is roughly equal to that in the ␤-␥ bridge oxygen. This indicates that, in the transition state, Ras shifts one-half of the negative charge that arises from P ␥-O␤-␥ fission from the ␤-␥ bridge oxygen to the two ␤ nonbridge oxygens. The KIE effects, interpreted in light of structural and spectroscopic data, suggest that Ras promotes a loose transition state by stabilizing negative charge in the ␤-␥ bridge and ␤ nonbridge oxygens of GTP. R as is the prototypical member of the family of small G proteins, which along with G␣ subunits of heterotrimeric G proteins, constitute a class of GTP hydrolases that regulate diverse signaling pathways in eukaryotes (1). Ras orchestrates multiple signaling pathways and regulates cell differentiation, proliferation, and apoptosis (2-4). The GTP-bound forms of G proteins are functionally active: that is, they bind to ''effector'' molecules and regulate their activities or location within the cell. Hydrolysis of GTP results in deactivation and effector release (5). In the absence of other factors, the duration of the active signaling state depends on the intrinsic hydrolytic rate of the G protein, which is typically very slow. However, Ras and other G proteins are subject to specific regulation by GTPase-activating proteins (GAPs), which accelerate intrinsic hydrolytic rates by factors ranging from 10 to 10 5 . In particular, RasGAP increases the GTPase rate of Ras by a factor of 10 5 , from 10 Ϫ4 s Ϫ1 to 10 s Ϫ1 (6). Mutations that impair either intrinsic or GAP-facilitated GTPase activity leave Ras in a prolonged state of activation, which is responsible for its role in oncogenic diseases (7).Ras catalyzes the in-line attack of water on the ␥ phosphate of GTP with inversion of configuration (8). However, the nature of the transition state and the rate-limiting step of Ras-catalyzed GTP hydrolysis remain unclear (9-16). A phosphoryl transfer reaction may either proceed through a metaphosphate or a phosphorane intermediate, or by a concerted pathway (Fig. 1) (17, 18...