Search

To be fully active and participate in the metabolism of phosphorylated nucleotides, most nucleoside diphosphate kinases (NDPKs) have to assemble into stable hexamers. Here we studied the role played by six intersubunit salt bridges R80-D93 in the stability of NDPK from the pathogen Mycobacterium tuberculosis ( Mt). Mutating R80 into Ala or Asn abolished the salt bridges. Unexpectedly, compensatory stabilizing mechanisms appeared for R80A and R80N mutants and we studied them by biochemical and structural methods. The R80A mutant crystallized into space group I222 that is unusual for NDPK, and its hexameric structure revealed the occurrence at the trimer interface of a stabilizing hydrophobic patch around the mutation. Functionally relevant, a trimer of the R80A hexamer showed a remodeling of the binding site. In this conformation, the cleft of the active site is more open, and then active His117 is more accessible to substrates. H/D exchange mass spectrometry analysis of the wild type and the R80A and R80N mutants showed that the remodeled region of the protein is highly solvent accessible, indicating that equilibrium between open and closed conformations is possible. We propose that such equilibrium occurs in vivo and explains how bulky substrates access the catalytic His117.

Most oligomeric proteins become active only after assembly, but why oligomerization is required to support function is not well understood. Here, we address this question using the wild type (WT) and a destabilized mutant (D93N) of the hexameric nucleoside diphosphate kinase from the pathogen Mycobacterium tuberculosis (Mt-NDPK). The conformational dynamics and oligomeric states of each were analyzed during unfolding and/or folding by hydrogen/deuterium exchange mass spectrometry (HDX-MS) at peptide resolution and by additional biochemical techniques. We found that WT and D93N native hexamers present a stable core and a flexible periphery, the latter being more flexible for the destabilized mutant. Stable but inactive species formed during unfolding of D93N and folding of WT were characterized. For the first time, we show that both of these species are nativelike dimers, each of its monomers having a major subdomain folded, while a minor subdomain (Kpn/α 0 ) remains unfolded. The Kpn/α 0 subdomain, which belongs to the catalytic site, becomes structured only upon hexamerization, explaining why oligomerization is required for NDPK activity. Further HDX-MS studies are necessary to establish the general activation mechanism for other homo-oligomers.

The mitochondrial ADP/ATP carrier is the paradigm of the mitochondrial carrier family (MCF), whose members are crucial for cross-talks between mitochondria, where cell energy is mainly produced, and the cytosol, where cell energy is mainly consumed. These carriers share structural and functional characteristics. Resolution of the 3D structure of the beef mitochondrial ADP/ATP carrier, in a complex with one of its specific inhibitors, revealed interesting features and suggested the involvement of some particular residues in substrate binding and transfer from the outside to the inside of mitochondria. To ascertain the role of these residues, namely, Y186, Y190, F191, and Y194, they were mutated into alanine in the yeast mitochondrial ADP/ATP carrier at equivalent positions (Y203, Y207, F208, and Y211). Two residues, Y203 and F208, appeared to be crucial for transport activity but not for substrate binding per se, indicating their involvement in the substrate transfer process through the carrier. Furthermore, it was possible to show that these mutations precluded conformational changes of the matrix loop m2, whose movements were demonstrated to participate in substrate transport by the wild-type carrier. Therefore, these aromatic residues may be involved in substrate gliding, and they may also confer specificity toward adenine nucleotides for the ADP/ATP carrier as compared with the MCF members.

The MraY transferase is an integral membrane protein that catalyzes an essential step of peptidoglycan biosynthesis, namely the transfer of the phospho-N-acetylmuramoyl-pentapeptide motif onto the undecaprenyl phosphate carrier lipid. It belongs to a large superfamily of eukaryotic and prokaryotic prenyl sugar transferases. No 3D structure has been reported for any member of this superfamily, and to date MraY is the only protein that has been successfully purified to homogeneity. Nineteen polar residues located in the five cytoplasmic segments of MraY appeared as invariants in the sequences of MraY orthologues. A certain number of these invariant residues were found to be conserved in the whole superfamily. To assess the importance of these residues in the catalytic process, site-directed mutagenesis was performed using the Bacillus subtilis MraY as a model. Fourteen residues were shown to be essential for MraY activity by an in vivo functional complementation assay using a constructed conditional mraY mutant strain. The corresponding mutant proteins were purified and biochemically characterized. None of these mutations did significantly affect the binding of the nucleotidic and lipidic substrates, but the k cat was dramatically reduced in almost all cases. The important residues for activity therefore appeared to be distributed in all the cytoplasmic segments, indicating that these five regions contribute to the structure of the catalytic site. Our data show that the D98 residue that is invariant in the whole superfamily should be involved in the deprotonation of the lipid substrate during the catalytic process.

Surfactant protein A (SP-A), a member of the collectin family that modulates innate immunity, has recently been involved in the physiology of reproduction. Consistent with the activation of ERK-1/2 and COX-2 induced by SP-A in myometrial cells, we reported previously the presence of two major proteins recognized by SP-A in these cells. Here we identify by mass spectrometry one of these SP-A targets as the intermediate filament (IF) desmin. In myometrial preparations derived from desmin-deficient mice, the absence of binding of SP-A to any 50 kDa protein confirmed the identity of this SP-A-binding site as desmin. Our data based on partial chymotrypsin digestion of pure desmin suggested that SP-A recognizes especially its rod domain, which is known to play an important role during the assembly of desmin into filaments. In line with that, electron microscopy experiments showed that SP-A inhibits in vitro the polymerization of desmin filaments. SP-A also recognized in vitro polymerized filaments in a calcium-dependent manner at a physiological ionic strength but not the C1q receptor gC1qR. Furthermore, Texas Red-labeled SP-A colocalized with desmin filaments in myometrial cells. Interestingly, vimentin, the IF characteristic of leukocytes, is one of the major proteins recognized by SP-A in protein extracts of U937 cells after PMA-induced differentiation of this monocytic cell line. Interaction of SP-A with vimentin was further confirmed using recombinant vimentin in solid-phase binding assays. The ability of SP-A to interact with desmin and vimentin, and to prevent polymerization of desmin monomers, shed light on unexpected and wider biological roles of this collectin.

The involvement of the b-subunit, subunit 4 in yeast, a component of the peripheral stalk of the ATP synthase, in the dimerization/oligomerization process of this enzyme was investigated. Increasing deletions were introduced by site-directed mutagenesis in the loop located in the mitochondrial intermembrane space and linking the two transmembrane (TM) segments of subunit 4. The resulting strains were still able to grow on nonfermentable media, but defects were observed in ATP synthase dimerization/oligomerization along with concomitant mitochondrial morphology alterations. Surprisingly, such defects, already depicted in the absence of the so-called dimer-specific subunits e and g, were found in a mutant harboring a full amount of subunit g associated to the monomeric form of the ATP synthase. Deletion of the intermembrane space loop of subunit 4 modified the profile of cross-linking products involving cysteine residues belonging to subunits 4, g, 6, and e. This suggests that this loop of subunit 4 participates in the organization of surrounding hydrophobic membranous components (including the two TM domains of subunit 4) and thus is involved in the stability of supramolecular species of yeast ATP synthase in the mitochondrial membrane.

It is now clearly established that dimerization of the F(1)F(o) ATP synthase takes place in the mitochondrial inner membrane. Interestingly, oligomerization of this enzyme seems to be involved in cristae morphogenesis. As they were able to form homodimers, subunits 4, e, and g have been proposed as potential ATP synthase dimerization subunits. In this paper, we provide evidence that subunit h, a peripheral stalk component, is located either at or near the ATP synthase dimerization interface. Subunit h homodimers were formed in mitochondria and were found to be associated to ATP synthase dimers. Moreover, homodimerization of subunit h and of subunit i turned out to be independent of subunits e and g, confirming the existence of an ATP synthase dimer in the mitochondrial inner membrane in the absence of subunits e and g. For the first time, this dimer has been observed by BN-PAGE. Finally, from these results we are now able to update our model for the supramolecular organization of the ATP synthase in the membrane and propose a role for subunits e and g, which stabilize the ATP synthase dimers and are involved in the oligomerization of the complex.

We have investigated the possibility of cell-fee synthesis of membrane proteins in the absence of a membrane and in the presence of detergent. We used the bacterial mechanosensitive channel MscL, a homopentamer, as a model protein. A wide range of nonionic or zwitterionic detergents, Triton X-100, Tween 20, Brij 58p, n-dodecyl beta-D-maltoside, and CHAPS, were compatible with cell-free synthesis, while n-octyl beta-D-glucoside and deoxycholate had an inhibitory effect. In vitro synthesis in the presence of Triton X-100 yielded milligram amounts of MscL per milliliter of lysate. Cross-linking experiments showed that the protein was able to oligomerize in detergents. When the purified protein was reconstituted in liposomes and studied by the patch-clamp technique, its activity at the single-molecule level was similar to that of the recombinant protein produced in Escherichia coli. Cell-free synthesis of membrane proteins should prove a valuable tool for the production of membrane proteins whose overexpression in heterologous systems is difficult.

Subunit h, a 92-residue-long, hydrophilic, acidic protein, is a component of the yeast mitochondrial F1Fo ATP synthase. This subunit, homologous to the mammalian factor F6, is essential for the correct assembly and/or functioning of this enzyme since yeast cells lacking it are not able to grow on nonfermentable carbon sources. Chemical cross-links between subunit h and subunit 4 have previously been shown, suggesting that subunit h is a component of the peripheral stalk of the F1Fo ATP synthase. The construction of cysteine-containing subunit h mutants and the use of bismaleimide reagents provided insights into its environment. Cross-links were obtained between subunit h and subunits alpha, f, d, and 4. These results and secondary structure predictions allowed us to build a structural model and to propose that this subunit occupies a central place in the peripheral stalk between the F1 sector and the membrane. In addition, subunit h was found to have a stoichiometry of one in the F1Fo ATP synthase complex and to be in close proximity to another subunit h belonging to another F1Fo ATP synthase in the inner mitochondrial membrane. Finally, functional characterization of mitochondria from mutants expressing different C-terminal shortened subunit h suggested that its C-terminal part is not essential for the assembly of a functional F1Fo ATP synthase.

The mitochondrial adenine nucleotide carrier, or Ancp, plays a key role in the maintenance of the energetic fluxes in eukaryotic cells. Human disorders have been found associated to unusual human ANC gene (HANC) expression but also to direct inactivation of the protein, either by autoantibody binding or by mutation. However, the individual biochemical properties of the three HAncp isoforms have not yet been deciphered. To do so, the three HANC ORF were expressed in yeast under the control of the regulatory sequences of ScANC2. Each of the three HANC was able to restore growth on a nonfermentable carbon source of a yeast mutant strain lacking its three endogenous ANC. Their ADP/ATP exchange properties could then be measured for the first time in isolated mitochondria. HANC3 was the most efficient to restore yeast growth, and HAnc3p presented the highest V(M) (80 nmol ADP min(-1) mg protein(-1)) and K(ADP)(M)(8.4 microM). HAnc1p and HAnc2p presented similar kinetic constants (V(M) approximately 30-40 nmol ADP min(-(1) mg protein(-1) and K(ADP)(M) approximately 2.5-3.7 microM), whose values were consistent with HANC1's and HANC2's lower capacity to restore yeast growth. However, the HANC genes restored growth at a lower level than ScANC2, indicating that HAncp amount may be limiting in vivo. To optimize the HAncp production, we investigated their biogenesis into mitochondria by mutagenesis of two charged amino acids in the N-terminus of HAnc1p. Severe effects were observed with the D3A and D3K mutations that precluded yeast growth. On the contrary, the K10A mutation increased yeast growth complementation and nucleotide exchange rate as compared to the wild type. These results point to the importance of the N-terminal region of HAnc1p for its biogenesis and transport activity in yeast mitochondria.

Cross-linking experiments showed that the supernumerary subunit i is close to the interface between two ATP synthases. These data were used to demonstrate the presence of ATP synthase dimers in the inner mitochondrial membrane of Saccharomyces cerevisiae. A cysteine residue was introduced into the inter-membrane space located C-terminal part of subunit i. Cross-linking experiments revealed a dimerization of subunit i. This cross-linking occurred only with the dimeric form of the enzyme after incubating intact mitochondria with a bis-maleimide reagent, thus indicating an inter-ATP synthase cross-linking, whereas the monomeric form of the enzyme exhibited only an intra-ATP synthase cross-linking with subunit 6, another component of the membranous domain of the ATP synthase.

FhuA (MM 78.9 kDa) is an Escherichia coli outer membrane protein that transports iron coupled to ferrichrome and is the receptor for a number of bacteriophages and protein antibiotics. Its three-dimensional structure consists of a 22-stranded beta-barrel lodged in the membrane, extracellular hydrophilic loops, and a globular domain (the "cork") located within the beta-barrel and occluding it. This unexpected structure raises questions about the connectivity of the different domains and their respective roles in the different functions of the protein. To address these questions, we have compared the properties of the wild-type receptor to those of a mutated FhuA (FhuA Delta) missing a large part of the cork. Differential scanning calorimetry experiments on wild-type FhuA indicated that the cork and the beta-barrel behave as autonomous domains that unfold at 65 and 75 degrees C, respectively. Ferrichrome had a strong stabilizing effect on the loops and cork since it shifted the first transition to 71.4 degrees C. Removal of the cork destabilized the protein since a unique transition at 61.6 degrees C was observed even in the presence of ferrichrome. FhuA Delta showed an increased sensitivity to proteolysis and to denaturant agents and an impairment in phage T5 and ferrichrome binding.

The topology of subunit i, a component of the yeast F(o)F(1)-ATP synthase, was determined by the use of cysteine-substituted mutants. The N(in)-C(out) orientation of this intrinsic subunit was confirmed by chemical modification of unique cysteine residues with 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid. Near-neighbor relationships between subunit i and subunits 6, f, g, and d were demonstrated by cross-link formation following sulfhydryl oxidation or reaction with homobifunctional and heterobifunctional reagents. Our data suggest interactions between the unique membrane-spanning segment of subunit i and the first transmembranous alpha-helix of subunit 6 and a stoichiometry of 1 subunit i per complex. Cross-linked products between mutant subunits i and proteins loosely bound to the F(o)F(1)-ATP synthase suggest that subunit i is located at the periphery of the enzyme and interacts with proteins of the inner mitochondrial membrane that are not involved in the structure of the yeast ATP synthase.

A component of the stator of the yeast ATP synthase (subunit 4 or b) showed many cross-linked products with the homobifunctional reagent dithiobis[succinimidyl propionate], which reacts with the amino group of lysine residues. The positions in subunit 4 that were involved in the cross-linkings were determined by using cysteine-generated mutants constructed by site-directed mutagenesis of ATP4. Cross-linking experiments with the heterobifunctional reagent p-azidophenacyl bromide, which has a spacer arm of 9 A, were performed with mitochondria and crude Triton X-100 extracts containing the solubilized enzyme. Substitution of lysine residues by cysteine residues in the hydrophilic C-terminal part of subunit 4 allowed cross-links with subunit h from C98 and with subunit d from C141, C143, and C151. OSCP was cross-linked from C174 and C209. A cross-linked product, 4+beta, was also obtained from C174. It is concluded that the C-terminus of subunit 4 is distant from the membrane surface and close to F(1) and OSCP. The N-terminal part of subunit 4 is close to subunit g, as demonstrated by the identification of a cross-linked product involving subunit g and the cysteine residues 7 or 14 of subunit 4.

Nucleoside diphosphate (NDP) kinases display low specificity with respect to the base moiety of the nucleotides and to the 2'-position of the ribose, but the 3'-hydroxyl is found to be important for catalysis. We report in this paper the enzymatic analysis of a series of derivatives of thymidine diphosphate (TDP) where the 3'-OH group was removed or replaced by fluorine, azido, and amino groups. With Dictyostelium NDP kinase, kcat decreases 15-200-fold from 1100 s-1 with TDP, and (kcat/Km)NDP decreases from 12 x 10(6) to 10(3) to 5 x 10(4) M-1 s-1, depending on the substrate. The poorest substrates are 3'-deoxyTDP and 3'-azido-3'-deoxyTDP, while the best modified substrates are 2',3'-dehydro-3'-deoxyTDP and 3'-fluoro-3'-deoxyTDP. In a similar way, 3'-fluoro-2',3'-dideoxyUDP was found to be a better substrate than 2',3'-dideoxyUDP, but a much poorer substrate than 2'-deoxyUDP. (kcat/Km)NDP is sensitive to the viscosity of the solution with TDP as the substrate but not with the modified substrates. To understand the poor catalytic efficiency of the modified nucleotides at a structural level, we determined the crystal structure of Dictyostelium NDP kinase complexed to 3'-fluoro-2',3'-dideoxyUDP at 2.7 A resolution. Significant differences are noted as compared to the TDP complex. Substrate-assisted catalysis by the 3'-OH, which is effective in the NDP kinase reaction, cannot occur with the modified substrate. With TDP, the beta-phosphate, which is the leaving group when a gamma-phosphate is transferred to His122, hydrogen bonds to the 3'-hydroxyl group of the sugar; with 3'-fluoro-2',3'-dideoxyUDP, the beta-phosphate hydrogen bonds to Asn119 and moves away from the attacking Ndelta of the catalytic His122. Since all anti-AIDS nucleoside drugs are modified at the 3'-position, these results are relevant to the role of NDP kinase in their cellular metabolism.

The arrangement of the N-terminal part of subunit 4 (subunit b) has been studied by the use of mutants containing cysteine residues in a loop connecting the two N-terminal postulated membrane-spanning segments. Labelling of the mutated subunit 4 by the fluorescent probe N-(7-(dimethylamino)-4-methyl-3-coumarinyl)maleimide revealed that the sulfhydryl groups were modified upon incubation of intact mitochondria. In addition, the nonpermeant sulfhydryl reagent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid prevented the 3-(N-maleimidylpropionyl)biocytin labeling of subunit 4D54C, thus showing a location of this residue in the intermembrane space. Cross-linking experiments revealed the proximity of subunits 4 and f. In addition a disulfide bridge between subunit 4D54C and subunit 6 was evidenced, thus demonstrating near-neighbor relationships of the two subunits and a location of the N-terminal part of the mitochondrially-encoded subunit 6 in the intermembrane space.

We introduced mutations to test the function of the hydrophobic sector of subunit 4 from Saccharomyces cerevisiae ATP synthase. Mutations were introduced at the chromosomic locus by homologous transformation of a strain disrupted in the ATP4 gene. The strain carrying the replacement Leu68-Val69-->Arg-Glu did not grow at 37 degrees C owing to a lack of assembly of F1 and Fo sectors at this temperature. The mutant strain grew slowly by oxidative phosphorylation at 28 degrees C with a growth yield 30% lower than the wild type. Analysis of the mutant strain showed a homogeneous population of altered ATP synthase with an energy coupling impairment. The mutant strain was oligomycin-resistant since the I50 value of oligomycin inhibition of ATPase and ATP synthase activities was 2-3-fold higher than that of the wild type, thus showing an alteration of the target to oligomycin. The level of phosphorylation or ATP induced a proton-dissipating pathway through Fo, which was insensitive to oligomycin but was sensitive to dicyclohexylcarbodiimide, thus suggesting an alteration in the regulation of ATP synthase proton permeability by the catalytic sector. From these results, we propose that the dicyclohexylcarbodiimide inhibition site is located upstream of the oligomycin inhibition site when considering the proton flux occurring during ATP synthesis.

This study investigates the relationships between the efficiency of oxidative phosphorylation (ATP/O) and respiratory flux in yeast mitochondria. To manipulate the electron flux through the respiratory chain, different substrates leading to NAD(P)H were used. By testing the effect of ADP either on respiratory rate in the presence or absence of oligomycin or on the level of NAD(P)H, on one hand, and the effects of uncouplers on respiration, on the other, we distinguished several categories of substrates: those for which the low respiration rate was mainly controlled by dehydrogenase activities and others for which the respiration was high and controlled downstream from the dehydrogenases. By using these different substrates, we observed that the ATP/O ratio decreased irrespective of the proton-motive force when the electron flux increased, unlike the situation when the respiratory rate was modulated by addition of the respiratory inhibitor. This result suggests that the oxidative phosphorylation efficiency depends on the value of the flux crossing the proton pumps. This relationship between efficiency (ATP/O) and electron flux was linked to a main control upstream from the respiratory chain. Such changes in the ATP/O ratio at least involved changes in the stoichiometry (H+/2e-) of the respiratory chain. Indeed, in non-phosphorylating mitochondria, the ratio of stoichiometries at site 2+3 over site 3 varied according to the proton-motive force. This cannot be explained by a variation in proton leak alone but involved both (i) a variable stoichiometry (H+/2e-) in relation to the electron flux value and (ii) different relationships between the variation in stoichiometry and the flux value at each coupling site.

Six affinity reagents containing chemically reactive groups, either on the phosphate residue at the 5'-end or on the 5'- or 3'-end internucleoside phosphate linkages of the oligothymidylate primers, were used to covalently modify the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). After covalent binding of these modified primer analogs to the enzyme, the addition of [alpha-32P]dTTP, in the presence of a complementary template, led to elongation of the primer. This reaction was catalyzed by the active site of the enzyme carrying the covalently bound primer. The relative efficiency of labeling of the p66/p51 heterodimer compared to the p66/p66 and p51/p51 homodimers of HIV-1 RT was in agreement with the previously determined affinity of the various enzyme forms toward different primers. The analogues preferentially modified the p66 subunit of the HIV-1 RT heterodimer. The labeling of all RT forms by synthetic primer analogues showed significant and specific competition by the natural primer of HIV-1 RT, tRNA(Lys). In addition, the kinetics of inactivation of RT by primer analogues was studied. The affinity of the enzyme to those derivatives in the presence of poly(A) template was about 5-10 times higher than in the absence of template. Moreover, the maximal rates of HIV-1 RT inactivation by analogues in the absence of template were 3-4 times higher. Our results suggest that the mechanism of oligonucleotide primer binding to HIV-1 RT is different in the presence or absence of template.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of a protonophoric uncoupler (CCCP) on the different cellular compartments was investigated in yeast grown aerobically on lactate. These cells were incubated in a resting cell medium under three conditions; in aerobiosis with lactate or glucose or in anaerobiosis with glucose as energetic substrate. For each condition, in vivo 31P NMR was used to measure pH gradients across vacuolar and plasma membrane and phosphorylated compound levels. Respiratory rate (aerobic conditions) and TPP+ uptake were measured independently. Concerning the polyphosphate metabolism, spontaneous NMR-detected polyphosphate breakdown occurred, in anaerobiosis and in the absence of CCCP. In contrast, in aerobiosis, polyphosphate hydrolysis was induced by addition of either CCCP or a vacuolar membrane ATPase-specific inhibitor, bafilomycin A1. Moreover, polyphosphates were totally absent in a null vacuolar ATPase activity mutant. The vacuolar polyphosphate content depended on two factors: vacuolar pH value, strictly linked to the vacuolar H(+)-ATPase activity, and inorganic phosphate concentration. CCCP was more efficient in dissipating the proton electrochemical gradient across vacuolar and mitochondrial membranes than across the plasma membrane. This discrepancy can be essentially explained by a difference of stimulability of each proton pump involved. As long as the energetic state (measured by NDP + NTP content) remains high, the plasma membrane proton ATPase is able to compensate the proton leak. Moreover, this ATPase contributes only partially to the generation of delta pH. The maintenance of the delta pH across the plasma membrane, that of the energetic state, and the cellular TPP+ uptake depend on the nature of the ATP-producing process.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of the amino acid sequence of beef pancreas tryptophanyl-tRNA synthetase was undertaken through both cDNA and direct peptide sequencing. A full-length cDNA clone containing a 475 amino acid open reading frame was obtained. The molecular mass of the corresponding peptide chain, 53,728 Da, was in agreement with that of beef tryptophanyl-tRNA synthetase, as determined by physicochemical methods (54 kDa). Expression of this clone in Escherichia coli led to tryptophanyl-tRNA synthetase activity in cell extracts. The open reading frame included two sequences analogous to the consensus sequences, HIGH and KMSKS, found in class I aminoacyl-tRNA synthetases. The homology with prokaryotic and yeast mitochondrial tryptophanyl-tRNA synthetases was low and was limited to the regions of the consensus sequences. However, a 90% homology was observed with the recently described rabbit peptide chain release factor (eRF) [Lee et al. (1990) Proc. Natl. Acad. Sci. 87, 3508-3512]. Such a strong homology may reveal a new group of genes deriving from a common ancestor, the products of which could be involved in tRNA aminoacylation (tryptophanyl-tRNA synthetase) or translation termination (eRF).

The promastigote surface protease (PSP) of Leishmania is a neutral membrane-bound zinc enzyme. The protease has no exopeptidase activity and does not cleave a large selection of substrates with chromogenic and fluorogenic leaving groups at the P1' site. The substrate specificity of the enzyme was studied by using natural and synthetic peptides of known amino acid sequence. The identification of 11 cleavage sites indicates that the enzyme preferentially cleaves peptides at the amino side when hydrophobic residues are in the P1' site and basic amino acid residues in the P2' and P3' sites. In addition, tyrosine residues are commonly found at the P1 site. Hydrolysis is not, however, restricted to these residues. These results have allowed the synthesis of a model peptide, H2N-L-I-A-Y-L-K-K-A-T-COOH, which is cleaved by PSP between the tyrosine and leucine residues with a kcat/Km ratio of 1.8 X 10(6) M-1 s-1. Furthermore, a synthetic nonapeptide overlapping the last four amino acids of the prosequence and the first five residues of mature PSP was found to be cleaved by the protease at the expected site to release the mature enzyme. This result suggests a possible autocatalytic mechanism for the activation of the protease. Finally, the hydroxamate-derivatized dipeptide Cbz-Tyr-Leu-NHOH was shown to inhibit PSP competitively with a KI of 17 microM.

The complete amino acid sequence of mature C8 beta has been derived from the DNA sequence of a cDNA clone identified by expression screening of a human liver cDNA library. Comparison with the amino acid sequence of C9 shows an overall homology with few deletions and insertions. In particular, the cysteine-rich domains and membrane-inserting regions of C9 are well conserved. These findings are discussed in relation to a possible mechanism of membrane attack complex formation.

A cDNA clone that is closely related to the granule-associated serine proteases of cytolytic T lymphocytes (CTL), called granzymes A-F, was isolated from a CTL expression library. The encoded serine protease, granzyme G, shows 70%-89% nucleotide identities to the granzymes C-F and, like those, consists of 228 amino acids preceded by the short propeptide Glu-Glu and a 18 residue long signal peptide. Granzyme G was identified by amino-terminal sequence analysis as a correctly processed and sorted protein stored in lysosome-like granules. The phylogenetic history of the granzyme multigene family was reconstructed by two tree-making methods and by Southern blot analyses of human, rat, and mouse DNA. Our results indicate differences in the evolutionary pathway between these species. The murine granzymes C-G descended from a progenitor present at the time of mammalian radiation. Granzyme C branched off first after the primate-rodent split and was involved in a recombination event with granzyme B before the rat-mouse divergence. Granzymes D and E have diverged after the mouse-rat speciation. However, no experimental evidence for the existence of a granzyme C-D-E-F-G equivalent was found in humans, and loss of the ancestral gene in the primate lineage is discussed. In view of the species differences in the number of granzyme gene copies during recent evolution, we propose that the murine granzymes B-G play several distinct roles in CTL-mediated effector functions as a response to quite recent changes of the biochemical environment.

Tryptophanyl-tRNA synthetase catalyzed formation of Trp-tRNA(Trp) has been studied by mixing tRNA(Trp) with a preformed bis(tryptophanyl adenylate)-enzyme complex in the 0-60-ms time range, on a quenched-flow apparatus. Analyzing the data gives an association rate constant ka = (1.22 +/- 0.47) X 10(8) M-1 S-1, a dissociation rate constant kd = 143 +/- 73 S-1, and a dissociation constant Kd = 1.34 +/- 0.80 microM for tRNA(Trp). The maximum rate constant of tryptophan transfer to tRNA(Trp) is kt = 33 +/- 3 S-1. When starting the aminoacylation reaction with a mono(tryptophanyl adenylate)-enzyme complex, one obtains different kinetic profiles than when using a bis(tryptophanyl adenylate)-enzyme complex. Over a 0-400-ms time range, the monoadenylate-enzyme complex yields an apparent first-order reaction, while the bis-adenylate-enzyme complex yields a biphasic aminoacylation of tRNA(Trp). Analysis of Trp-tRNA(Trp) formation from both complexes according to simple reaction schemes shows that the dissociation of tRNA(Trp) from an enzyme subunit carrying no adenylate is 6.9-fold slower than from an enzyme subunit carrying an adenylate. The apparent rate constant of dissociation of nascent tryptophanyl-tRNA(Trp) is 4.9 S-1 in the absence of free tryptophan, which is much slower than its rate of formation (33 S-1).(ABSTRACT TRUNCATED AT 250 WORDS)