Structure and function of ubiquitin conjugating enzyme E2-25K: the tail is a core-dependent activity element

Individual members of the conserved family of ubiquitin conjugating enzymes (E2s) mediate the ubiquitination and turnover of specific substrates of the ubiquitin-dependent degradation pathway. E2 proteins have a highly conserved core domain of approximately 150 amino acids which contains the active-site Cys. Certain E2s have unique terminal extensions, which are thought to contribute to selective E2 function by interacting either with substrates or with trans-acting factors such as ubiquitin-protein ligases (E3s). We used the mammalian ubiquitin conjugating enzyme E2-25K in a biochemical test of this hypothesis. The properties of two truncated derivatives show that the 47-residue tail of E2-25K is necessary for three of the enzyme's characteristic properties: high activity in the synthesis of unanchored K48-linked polyubiquitin chains; resistance of the active-site Cys residue to alkylation; and an unusual discrimination against noncognate (nonmammalian) ubiquitin activating (E1) enzymes. However, the tail is not sufficient to generate these properties, as shown by the characteristics of a chimeric enzyme in which the tail of E2-25K was fused to the core domain of yeast UBC4. These and other results indicate that the specific biochemical function of the tail is strongly dependent upon unique features of the E2-25K core domain. Thus, divergent regions within the conserved core domains of E2 proteins may be highly significant for function. Expression of truncated E2-25K as a glutathione S-transferase (GST) fusion protein resulted in the apparent recovery of E2-25K-specific properties, including activity in chain synthesis. However, the catalytic mechanism utilized by the truncated fusion protein proved to be distinct from the mechanism utilized by the wild-type enzyme. The unexpected properties of the fusion protein were due to GST-induced dimerization. These results indicate the potential for self-association to modulate the polyubiquitin chain synthesis activities of E2 proteins, and indicate that caution should be applied in interpreting the activities of GST fusion proteins.     

Crystal structure of the human ubiquitin-like protein NEDD8 and interactions with ubiquitin pathway enzymes

The NEDD8/Rub1 class of ubiquitin-like proteins has been implicated in progression of the cell cycle from G1 into S phase. These molecules undergo a metabolism that parallels that of ubiquitin and involves specific interactions with many different proteins. We report here the crystal structure of recombinant human NEDD8 refined at 1.6-A resolution to an R factor of 21.9%. As expected from the high sequence similarity (57% identical), the NEDD8 structure closely resembles that reported previously for ubiquitin. We also show that recombinant human NEDD8 protein is activated, albeit inefficiently, by the ubiquitin-activating (E1) enzyme and that NEDD8 can be transferred from E1 to the ubiquitin conjugating enzyme E2-25K. E2-25K adds NEDD8 to a polyubiquitin chain with an efficiency similar to that of ubiquitin. A chimeric tetramer composed of three ubiquitins and one histidine-tagged NEDD8 binds to the 26 S proteasome with an affinity similar to that of tetraubiquitin. Seven residues that differ from the corresponding residues in ubiquitin, but are conserved between NEDD8 orthologs, are candidates for mediating interactions with NEDD8-specific partners. One such residue, Ala-72 (Arg in ubiquitin), is shown to perform a key role in selecting against reaction with the ubiquitin E1 enzyme, thereby acting to prevent the inappropriate diversion of NEDD8 into ubiquitin-specific pathways.     

Activity of ubiquitin-dependent pathway in response to oxidative stress. Ubiquitin-activating enzyme is transiently up-regulated

Relations between the ubiquitin pathway and cellular stress have been noted, but data regarding responses of the ubiquitin pathway to oxidative stress are scanty. This paper documents the response of this pathway to oxidative stress in lens cells. A brief exposure of lens epithelial cells to physiologically relevant levels of H2O2 induces a transient increase in activity of the ubiquitin-dependent pathway. Ubiquitin conjugation activity was maximal and increased 3. 5-9.2-fold over the activity noted in untreated cells by 4 h after removal of H2O2. By 24 h after removal of H2O2, ubiquitin conjugation activity returned to the level noted in untreated cells. In parallel to the changes in ubiquitin conjugation activity, the activity of ubiquitin-activating enzyme (E1), as determined by thiol ester formation, increased 2-6.7-fold during recovery from oxidation. Addition of exogenous E1 resulted in an increase in ubiquitin conjugation activity and in the levels of ubiquitin carrier protein (E2)-ubiquitin thiol esters in both the untreated cells and the H2O2-treated cells. These data suggest that E1 is the rate-limiting enzyme in the ubiquitin conjugation process and that the increases in ubiquitin conjugation activity which are induced upon recovery from oxidation are primarily due to increased E1 activity. The oxidation- and recovery-induced up-regulation of E1 activity is primarily due to post-synthetic events. Substrate availability and up-regulation of E2 activities also appear to be related to the enhancement in ubiquitinylation upon recovery from oxidative stress. The oxidation-induced increases in ubiquitin conjugation activity were associated with an increase in intracellular proteolysis, suggesting that the transient increase in ubiquitinylation noted upon recovery from oxidative stress may play a role in removal of damaged proteins from the cells.     

Role of active site tyrosine residues in catalysis by human glutathione reductase

Tyr114 and Tyr197 are highly conserved residues in the active site of human glutathione reductase, Tyr114 in the glutathione disulfide (GSSG) binding site and Tyr197 in the NADPH site. Mutation of either residue has profound effects on catalysis. Y197S and Y114L have 17% and 14% the activity of the wild-type enzyme, respectively. Mutation of Tyr197, in the NADPH site, leads to a decrease in Km for GSSG, and mutation of Tyr114, in the GSSG site, leads to a decrease in Km for NADPH. This behavior is predicted for enzymes operating by a ping-pong mechanism where both half-reactions partially limit turnover. Titration of the wild-type enzyme or Y114L with NADPH proceeds in two phases, Eox to EH2 and EH2 to EH2-NADPH. In contrast, Y197S reacts monophasically, showing that excess NADPH fails to enhance the absorbance of the thiolate-FAD charge-transfer complex, the predominant EH2 form of glutathione reductase. The reductive half-reactions of the wild-type enzyme and of Y114L are similar; FAD reduction is fast (approximately 500 s-1 at 4 degreesC) and thiolate-FAD charge-transfer complex formation has a rate of 100 s-1. In Y197S, these rates are only 78 and 5 s-1, respectively. The oxidative half-reaction, the rate of reoxidation of EH2 by GSSG, of the wild-type enzyme is approximately 4-fold faster than that of Y114L. These results are consistent with Tyr197 serving as a gate in the binding of NADPH, and they indicate that Tyr114 assists the acid catalyst His467'.     

Mutations in the activation loop tyrosine of the oncoprotein v-Fps

Mutations were made in the activation loop tyrosine of the kinase domain of the oncoprotein v-Fps to assess the role of autophosphorylation in catalysis. Three mutant proteins, Y1073E, Y1073Q, and Y1073F, were expressed and purified as fusion proteins of glutathione-S-transferase from Escherichia coli and their catalytic properties were evaluated. Y1073E, Y1073Q, and Y1073F have k(cat) values that are reduced by 5-, 35-, and 40-fold relative to the wild-type enzyme, respectively. For all mutant enzymes, the Km values for ATP and a peptide substrate, EAEIYEAIE, are changed by 0.4-2-fold compared to the wild-type enzyme. The slopes for the plots of relative turnover versus solvent viscosity [(k(cat))eta] are 0.71 +/- 0.08, 0.10 +/- 0.06, and approximately 0 for wild type, Y1073Q, and Y1073E, respectively. These results imply that the phosphoryl transfer rate constant is reduced by 19- and 130-fold for Y1073E and Y1073Q compared to the wild-type enzyme. The dissociation constant of the substrate peptide is 1.5-2.5-fold lower for the mutants compared to wild type. The inhibition constant for EAEIFEAIE, a competitive inhibitor, is unaffected for Y1073E and raised 3-fold for Y1073Q compared to wild type. Y1073E and Y1073Q are strongly activated by free magnesium to the same extent and the apparent affinity constant for the metal is similar to that for the wild-type enzyme. The data indicate that the major role of autophosphorylation in the tyrosine kinase domain of v-Fps is to increase the rate of phosphoryl transfer without greatly affecting active-site accessibility or the local environment of the activating metal. Finally, the similar rate enhancements for phosphoryl transfer in v-Fps compared to protein kinase A [Adams et al. (1995) Biochemistry 34, 2447-2454] upon autophosphorylation suggest a conserved mechanism for communication between the activation loop and the catalytic residues of these two enzymes.     

Effect of CTP synthetase regulation by CTP on phospholipid synthesis in Saccharomyces cerevisiae

CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) activity in Saccharomyces cerevisiae is allosterically regulated by CTP product inhibition. Amino acid residue Glu161 in the URA7-encoded and URA8-encoded CTP synthetases was identified as being involved in the regulation of these enzymes by CTP product inhibition. The specific activities of the URA7-encoded and URA8-encoded enzymes with a Glu161 --> Lys (E161K) mutation were 2-fold greater when compared with the wild-type enzymes. The E161K mutant URA7-encoded and URA8-encoded CTP synthetases were less sensitive to CTP product inhibition with inhibitor constants for CTP of 8.4- and 5-fold greater, respectively, than those of their wild-type counterparts. Cells expressing the E161K mutant enzymes on a multicopy plasmid exhibited an increase in resistance to the pyrimidine poison and cancer therapeutic drug cyclopentenylcytosine and accumulated elevated (6-15-fold) levels of CTP when compared with cells expressing the wild-type enzymes. Cells expressing the E161K mutation in the URA7-encoded CTP synthetase exhibited an increase (1.5-fold) in the utilization of the Kennedy pathway for phosphatidylcholine synthesis when compared with control cells. Cells bearing the mutation also exhibited an increase in the synthesis of phosphatidylcholine (1.5-fold), phosphatidylethanolamine (1.3-fold), and phosphatidate (2-fold) and a decrease in the synthesis of phosphatidylserine (1.7-fold). These alterations were accompanied by an inositol excretion phenotype due to the misregulation of the INO1 gene. Moreover, cells bearing the E161K mutation exhibited an increase (1.6-fold) in the ratio of total neutral lipids to phospholipids, an increase in triacylglycerol (1.4-fold), free fatty acids (1.7-fold), and ergosterol ester (1.8-fold), and a decrease in diacylglycerol (1. 3-fold) when compared with control cells. These data indicated that the regulation of CTP synthetase activity by CTP plays an important role in the regulation of phospholipid synthesis.     

Site-directed mutagenesis of human leukotriene C4 synthase

The functional characteristics of leukotriene C4 synthase (LTC4S), which specifically conjugates leukotriene A4 with GSH, were assessed by mutagenic analysis. Human LTC4S and the 5-lipoxygenase-activating protein share substantial amino acid identity and predicted secondary structure. The mutation of Arg-51 of LTC4S to Thr or Ile abolishes the enzyme function, whereas the mutation of Arg-51 to His or Lys provides a fully active recombinant protein. The mutations Y59F, Y97F, Y93F, N55A, V49F, and A52S increase the Km of the recombinant microsomal enzyme for GSH. The mutation Y93F also markedly reduces enzyme function and increases the optimum for pH-dependent activity. The deletion of the third hydrophobic domain with the carboxyl terminus abolishes the enzyme activity, and function is restored by the substitution of the third hydrophobic domain and carboxyl terminus of 5-lipoxygenase-activating protein for that of LTC4S. Mutations of C56S and C82V alone or together and the deletion of Lys-2 and Asp-3 of LTC4S do not alter enzyme function. The direct linkage of two LTC4S monomers by a 12-amino acid bridge provides an active dimer, and the same bridging of inactive R51I with a wild-type monomer creates an active pseudo-dimer with function similar to that of the wild-type enzyme. These results suggest that in the catalytic function of LTC4S, Arg-51 probably opens the epoxide ring and Tyr-93 provides the thiolate anion of GSH. Furthermore, the monomer has independent conjugation activity, and dimerization of LTC4S maintains the proper protein structure.     

Probing the roles of active site residues in phosphatidylinositol-specific phospholipase C from Bacillus cereus by site-directed mutagenesis

The role of amino acid residues located in the active site pocket of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus[Heinz, D. W., Ryan, M., Bullock, T., & Griffith, O. H. (1995) EMBO J. 14, 3855-3863] was investigated by site-directed mutagenesis, kinetics, and crystal structure analysis. Twelve residues involved in catalysis and substrate binding (His32, Arg69, His82, Gly83, Lys115, Glu117, Arg163, Trp178, Asp180, Asp198, Tyr200, and Asp274) were individually replaced by 1-3 other amino acids, resulting in a total number of 21 mutants. Replacements in the mutants H32A, H32L, R69A, R69E, R69K, H82A, H82L, E117K, R163I, D198A, D198E, D198S, Y200S, and D274S caused essentially complete inactivation of the enzyme. The remaining mutants (G83S, K115E, R163K, W178Y, D180S, Y200F, and D274N) exhibited reduced activities up to 57% when compared with wild-type PI-PLC. Crystal structures determined at a resolution ranging from 2.0 to 2.7 A for six mutants (H32A, H32L, R163K, D198E, D274N, and D274S) showed that significant changes were confined to the site of the respective mutation without perturbation of the rest of the structure. Only in mutant D198E do the side chains of two neighboring arginine residues move across the inositol binding pocket toward the newly introduced glutamic acid. An analysis of these structure-function relationships provides new insight into the catalytic mechanism, and suggests a molecular explanation of some of the substrate stereospecificity and inhibitor binding data available for this enzyme.     

Active site of 5-aminolevulinate synthase resides at the subunit interface. Evidence from in vivo heterodimer formation

5-Aminolevulinate synthase (EC 2.3.1.37) is the first enzyme in the heme biosynthetic pathway of animals, fungi and some bacteria. It functions as a homodimer and requires pyridoxal 5'-phosphate as an essential cofactor. In mouse erythroid 5-aminolevulinate synthase, lysine 313 has been identified as the residue involved in the Schiff base linkage with pyridoxal 5'-phosphate [Ferreira, G. C., et al. (1993) Protein Sci. 2, 1959-1965], while arginine 149, a conserved residue among all known 5-aminolevulinate synthase sequences, is essential for function [Gong & Ferreira (1995) Biochemistry 34, 1678-1685]. To determine whether each subunit contains an independent active site (i.e., intrasubunit arrangement) or whether the active site resides at the subunit interface (i.e., intersubunit arrangement), in vivo complementation studies were used to generate heterodimers from site-directed, catalytically inactive mouse 5-aminolevulinate synthase mutants. When R149A and K313A mutants were co-expressed in a hem A- Escherichia coli strain, which can only grow in the presence of 5-aminolevulinate or when it is transformed with an active 5-aminolevulinate synthase expression plasmid, the hem A- E. coli strain acquired heme prototrophy. The purified K313A/R149A heterodimer mixture exhibited K(m) values for the substrates similar to those of the wild-type enzyme and approximately 26% of the wild-type enzyme activity which is in agreement with the expected 25% value for the K313A/R149A coexpression system. In addition, DNA sequencing of four Saccharomyces cerevisiae 5-aminolevulinate synthase mutants, which lack ALAS activity but exhibit enzymatic complementation, revealed that mutant G101 with mutations N157Y and N162S can complement mutant G220 with mutation T452R, and mutant G205 with mutation C145R can complement mutant Ole3 with mutation G344C. Taken together, these results provide conclusive evidence that the 5-aminolevulinate synthase active site is located at the subunit interface and contains catalytically essential residues from the two subunits.     

Identification of amino acid residues in a class I ubiquitin-conjugating enzyme involved in determining specificity of conjugation of ubiquitin to proteins

The ubiquitin pathway is a major system for selective proteolysis in eukaryotes. However, the mechanisms underlying substrate selectivity by the ubiquitin system remain unclear. We previously identified isoforms of a rat ubiquitin-conjugating enzyme (E2) homologous to the Saccharomyces cerevisiae class I E2 genes, UBC4/UBC5. Two isoforms, although 93% identical, show distinct features. UBC4-1 is expressed ubiquitously, whereas UBC4-testis is expressed in spermatids. Interestingly, although these isoforms interacted similarly with some ubiquitin-protein ligases (E3s) such as E6-AP and rat p100 and an E3 that conjugates ubiquitin to histone H2A, they also supported conjugation of ubiquitin to distinct subsets of testis proteins. UBC4-1 showed an 11-fold greater ability to support conjugation of ubiquitin to endogenous substrates present in a testis nuclear fraction. Site-directed mutagenesis of the UBC4-testis isoform was undertaken to identify regions of the molecule responsible for the observed difference in substrate specificity. Four residues (Gln-15, Ala-49, Ser-107, and Gln-125) scattered on surfaces away from the active site appeared necessary and sufficient for UBC4-1-like conjugation. These four residues identify a large surface of the E2 core domain that may represent an area of binding to E3s or substrates. These findings demonstrate that a limited number of amino acid substitutions in E2s can dictate conjugation of ubiquitin to different proteins and indicate a mechanism by which small E2 molecules can encode a wide range of substrate specificities.     

Macrolides and drug interactions: review of the literature]

Annexin I is a member of the annexin family of calcium-dependent membrane binding proteins. The core domain of these proteins is similar in all annexins but the N-terminal domain is specific for each member. This domain is thought to regulate annexin function through phosphorylation. In annexin I, Ser-27 is one of the amino acids that can be phosphorylated by protein kinase C. Phosphorylations are thought to regulate some annexin I functions by increasing calcium requirement. Two mutants were prepared in this study: S27E and S27A proteins. The first mimics phosphorylation while the second prevents phosphorylation at residue 27. Wild-type annexin I and S27A mutant protein showed the same calcium dependence for phospholipid vesicles aggregation, while S27E mutant protein showed a higher calcium requirement and a low maximal extent of aggregation. By contrast, liposome binding and self-association required identical calcium concentrations for the wild-type and the two mutant proteins. To examine whether the regulation observed is due to modification of the N-terminal structure, we investigated conformational changes by using two approaches. Firstly we analysed proteinase sensibility. Limited proteolysis of the N-terminal tail showed similar patterns for the three proteins. Using drastic conditions of proteolysis, we observed strong resistance of the core domain to digestion in the presence of calcium. Secondly, since Ser-27 is located on the N-terminal domain that contains a tryptophan located at position 12, the fluorescence of this residue was analysed during Ca2+-binding of wild-type annexin I and S27E mutant protein. The results demonstrated that Ca2+ induces a slight change in the Trp environment of wild-type annexin I, corresponding to a burying of the residue. No changes in fluorescence features were observed with S27E mutant protein. The results obtained show that phosphorylation of the N-terminal tail plays a regulatory role in phospholipid vesicle aggregation, which is based on a mechanism distinct from protein self-association. This phosphorylation induces a conformational change in the tail probably related to aggregation property.     

A two-year prospective study of the effect of ursodeoxycholic acid on urinary bile acid excretion and liver morphology in cystic fibrosis-associated liver disease

Mice constitutively express glutathione S-transferase mGSTA3-3 in liver. This isoform possesses uniquely high conjugating activity toward aflatoxin B1-8,9-epoxide (AFBO), thereby protecting mice from aflatoxin B1-induced hepatocarcinogenicity. In contrast, rats constitutively express a closely related GST isoenzyme, rGSTA3-3, with low AFBO activity and, therefore, are sensitive to aflatoxin B1 exposure. Although the two GSTs share 86% sequence identity and have similar catalytic activities toward 1-chloro-2,4-dinitrobenzene (CDNB), they have an approximately 1000-fold difference in catalytic activity toward AFBO. To identify amino acids that confer high activity toward AFBO, non-conserved rGSTA3-3 residues were replaced with mGSTA3-3 residues in two regions believed to form the substrate binding site. Twenty-one mutant rGSTA3-3 enzymes were generated by site-directed mutagenesis using combinations of nine different residues. Except for the E208D mutant, single mutations of rGSTA3-3 produced enzymes with no detectable AFBO activity. Generally, AFBO conjugation activity increased in additive fashion as mGSTA3-3 residues were introduced into the rGSTA3-3 enzyme with the six site mutant E104I/H108Y/Y111H/L207F/E208D/V217K displaying the highest AFBO activity (40 nmol/mg/min) of all the mutant enzymes. When this mutant enzyme was further modified by three additional substitutions (D103E/I105M/V106I) AFBO conjugation activity decreased 14-fold to 2. 8 nmol/mg/min. Although wild-type mGSTA3-3 AFBO conjugation activity (265 nmol/mg/min) could not be obtained by our rGSTA3-3 mutants, we were able to identify six mGSTA3-3 residues; Ile104, Tyr108, His111, Phe207, Asp208, and Lys217 that, when collectively substituted into rGSTA3-3, substantially increased (>200-fold) glutathione conjugation activity toward AFBO.     

Essential role of E2-25K/Hip-2 in mediating amyloid-beta neurotoxicity

The ubiquitin/proteasome system has been proposed to play an important role in Alzheimer's disease (AD) pathogenesis. However, the critical factor(s) modulating both amyloid-beta peptide (Abeta) neurotoxicity and ubiquitin/proteasome system in AD are not known. We report the isolation of an unusual ubiquitin-conjugating enzyme, E2-25K/Hip-2, as a mediator of Abeta toxicity. The expression of E2-25K/Hip-2 was upregulated in the neurons exposed to Abeta(1-42) in vivo and in culture. Enzymatic activity of E2-25K/Hip-2 was required for both Abeta(1-42) neurotoxicity and inhibition of proteasome activity. E2-25K/Hip-2 functioned upstream of apoptosis signal-regulating kinase 1 (ASK1) and c-Jun N-terminal kinase (JNK) in Abeta(1-42) toxicity. Further, the ubiquitin mutant, UBB+1, a potent inhibitor of the proteasome which is found in Alzheimer's brains, was colocalized and functionally interacted with E2-25K/Hip-2 in mediating neurotoxicity. These results suggest that E2-25K/Hip-2 is a crucial factor in regulating Abeta neurotoxicity and could play a role in the pathogenesis of Alzheimer's disease.     

Ectopic expression of inactive forms of yeast DNA topoisomerase II confers resistance to the anti-tumour drug, etoposide

Drug resistance to anti-tumour agents often coincides with mutations in the gene encoding DNA topoisomerase II alpha. To examine how inactive forms of topoisomerase II can influence resistance to the chemotherapeutic agent VP-16 (etoposide) in the presence of a wild-type allele, we have expressed point mutations and carboxy-terminal truncations of yeast topoisomerase II from a plasmid in budding yeast. Truncations that terminate the coding region of topoisomerase II at amino acid (aa) 750, aa 951 and aa 1044 are localised to both the cytosol and the nucleus and fail to complement a temperature-sensitive top2-1 allele at non-permissive temperature. In contrast, the plasmid-borne wild-type TOP2 allele and a truncation at aa 1236 are nuclear localised and complement the top2-1 mutation. At low levels of expression, truncated forms of topoisomerase II render yeast resistant to levels of etoposide 2- and 3-fold above that tolerated by cells expressing the full-length enzyme. Maximal resistance is conferred by the full-length enzyme carrying a mutated active site (Y783F) or a truncation at aa 1044. The level of phosphorylation of topoisomerase II was previously shown to correlate with drug resistance in cultured cells, hence we tested mutants in the major casein kinase II acceptor sites in the C-terminal domain of yeast topoisomerase II for changes in drug sensitivity. Neither ectopic expression of the C-terminal domain alone nor phosphoacceptor site mutants significantly alter the host cell's sensitivity to etoposide.     

Toxicological effects of beryllium on platelets and vascular endothelium

The crystal structure of rabbit muscle pyruvate kinase complexed with Mn2+, K+, and pyruvate revealed a binding site of K+ [T. M. Larsen, L. T. Laughlin, H. M. Holden, I. Rayment, and G. H. Reed (1994) Biochemistry 33, 6301-6309]. Sequence comparisons of rabbit muscle pyruvate kinase and pyruvate kinases from Corynebacterium glutamicum and Escherichia coli, which do not exhibit a requirement for activation by monovalent cations, indicate that the only substitutions in the K+ binding site are conservative. Glu 117 in the rabbit muscle enzyme, which is close to the K+ site, is, however, replaced by Lys in these two bacterial pyruvate kinases. The proximity of Glu 117 to K+ in the structure of the rabbit enzyme and conservation of the binding site in the bacterial enzymes which lack a dependence on monovalent cations suggested that a protonated epsilon-amino group of Lys 117 in these bacterial enzymes may provide an "internal monovalent cation." Site-specific mutant forms of the rabbit enzyme corresponding to E117K, E117A, E117D, and E117K/K114Q pyruvate kinase were examined to test this hypothesis. The E117K pyruvate kinase exhibits 12% of the activity of the fully activated wild-type enzyme but is > 200-fold more active than the wild-type enzyme in the absence of activating monovalent cations. Moreover, the activity of E117K pyruvate kinase exhibits no stimulation by monovalent cations in the assay mixtures. Both E117A and E117D pyruvate kinases retain activation by monovalent cations but have reduced activities relative to wild type. The results are consistent with the hypothesis that pyruvate kinases that do not require activation by monovalent cations supply an internal monovalent cation in the form of a protonated epsilon-amino group of Lys. The results also support the assignment of the monovalent cation in the active site of pyruvate kinase.     

The role of tyrosine-114 in the enzymatic activity of the Shiga-like toxin I A-chain

Shiga-like toxin I (SLT-I), the potent cytotoxin produced by certain pathogenic strains of Escherichia coli, is a member of a burgeoning family of ribosome-in-activating proteins (RIPs), which share common structural and mechanistic features. The prototype of the group is the plant toxin ricin. Recently we proposed a structural model for the Slt-IA active site, based in part on the known geometry of the enzymatic subunit of the ricin toxin. The model places three aromatic residues within the putative Slt-IA active site cleft: tyrosine 77, tyrosine 114, and tryptophan 203. Here we present biochemical and biophysical data regarding, the phenotypes of conservative point mutants of Slt-IA in which tyrosine 114 is altered. We used oligonucleotide-directed mutagenesis to replace tyrosine 114 with either phenylalanine (Y114F) or serine (Y114S). Periplasmic extracts of E. coli containing wild-type or mutant Slt-IA were tested for their ability to inhibit protein synthesis in vitro. Relative to wild-type, the activity of mutant Y114F was attenuated about 30-fold, while the mutant Y114S was attenuated about 500 to 1000-fold. In order to address the possibility that differential activation of the mutants rather than local effects at the active site might account for their diminished activity, we engineered the same mutations into a truncated slt-IA cassette that directs expression of a product corresponding to the activated A1 form of Slt-IA (wild-type-delta). The same general relationships held: relative to wild type-delta, Y114F-delta was attenuated about 7-fold, and Y114S-delta about 300-fold.(ABSTRACT TRUNCATED AT 250 WORDS)     

High affinity binding of TAR RNA by the human immunodeficiency virus type-1 tat protein requires base-pairs in the RNA stem and amino acid residues flanking the basic region

The binding site for tat protein on TAR RNA has been defined in quantitative terms using an extensive series of mutations. The relative dissociation constants for the mutant TAR RNAs were measured using a dual-label competition filter binding assay in which 35S-labelled wild-type TAR RNA (K1) was competed against 3H-labelled mutant TAR RNA (K2). The error in the self-competition experiment was usually less than 10% (e.g. K2/K1 = 1.07 +/- 0.05, n = 19) and the experimental data accurately matched theoretical curves calculated with fitted dissociation constants. Mutations in U23, a critical residue in the U-rich "bulge" sequence, or in either of the two base-pairs immediately above the "bulge", G26.C39 and A27.U38 reduced that affinity by 8- to 20-fold. Significant contributions to tat binding affinity were also made by the base-pairs located immediately below the bulge. For example, mutation of A22.U40 to U.A reduced tat affinity 5-fold, and mutation of G21.C41 to C.G reduced tat affinity 4-fold. The binding of a series of peptides spanning the basic "arginine-rich" sequence of tat was examined using both filter-binding and gel mobility shift assays. Each of the peptides showed significantly reduced affinities for wild-type TAR RNA compared to the tat protein. The ADP-2 (residues 43 to 72), ADP-3 (residues 48 to 72) and ADP-5 (residues 49 to 86) peptides were unable to discriminate between wild-type TAR RNA and TAR RNA mutants with the same fidelity as the tat protein. For example, these peptides showed no more than 3-fold reductions in affinity relative to wild-type TAR RNA for the U23-->C mutation in the bulge, or G26.G39-->C.G mutation in the stem of TAR RNA. By contrast, the ADP-I (residues 37 to 72), ADP-4 (residues 32 to 62) and ADP-6 (residues 32 to 72) peptides, which each carry amino acid residues from the "core" region of the tat protein have binding specificities that more closely resemble the protein. The ADP-4 and ADP-6 peptides showed between 4- and 7-fold reductions in affinity for the U23-->C or G26.C39-->C.G mutations. The ADP-1 peptide most closely resembles the protein in its binding specificity and showed 9-fold and 14-fold reductions in affinity for the two mutants, respectively. Chemical-modification interference assays using diethylpyrocarbonate (DEPC) and ethylnitrosourea (ENU) were also used to compare the binding properties of the tat protein and the tat-derived peptides.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of Asp258 as the metal coordinate of pigeon liver malic enzyme by site-specific mutagenesis

Pigeon liver malic enzyme was inactivated by ferrous sulfate in the presence of ascorbate. Manganese and some other divalent metal ions provided complete protection of the enzyme against the Fe(2+)-induced inactivation. The inactivated enzyme was subsequently cleaved by the Fe(2+)-ascorbate system at Asp258-Ile259, which was presumably the Mn(2+)-binding site of the enzyme [Wei, C. H., Chou, W. Y., Huang, S. M., Lin, C. C., & Chang, G. G. (1994) Biochemistry 33, 7793-7936]. For identification of Asp258 as the putative metal-binding site of the enzyme, we prepared four mutant enzymes substituted at Asp258 with glutamate (D258E), asparagine (D258N), lysine (D258K), or alanine (D258A), respectively. These mutant proteins were recombinantly expressed in a bacterial expression system (pET-15b) with a stretch of histidine residues attached at the N-terminus and were successfully purified to apparent homogeneity by a single Ni-chelated affinity column. Among the four mutants, only D258E possessed 0.8% residual activity after purification; all other purified mutants had < 0.0001% residual activity in catalyzing the oxidative decarboxylation of L-malate. The D258E mutant was susceptible to inactivation by the Fe(2+)-ascorbate system, albeit with much slower inactivation rate, and was protected by the Mn2+ to a lesser extent as compared to the wild-type enzyme. None of the mutants were cleaved by the Fe(2+)-ascorbate system under conditions that cleaved the natural or wild-type enzyme at Asp258.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tryptophan synthase mutations that alter cofactor chemistry lead to mechanism-based inactivation

Mutations in the pyridoxal phosphate binding site of the tryptophan synthase beta subunit (S377D and S377E) alter cofactor chemistry [Jhee, K.-H., et al. (1998) J. Biol. Chem. 273, 11417-11422]. We now report that the S377D, S377E, and S377A beta2 subunits form alpha2 beta2 complexes with the alpha subunit and activate the alpha subunit-catalyzed cleavage of indole 3-glycerol phosphate. The apparent Kd for dissociation of the alpha and beta subunits is unaffected by the S377A mutation but is increased up to 500-fold by the S377D and S377E mutations. Although the three mutant alpha2 beta2 complexes exhibit very low activities in beta elimination and beta replacement reactions catalyzed at the beta site in the presence of Na+, the activities and spectroscopic properties of the S377A alpha2 beta2 complex are partially repaired by addition of Cs+. The S377D and S377E alpha2 beta2 complexes, unlike the wild-type and S377A alpha2 beta2 complexes and the mutant beta2 subunits, undergo irreversible substrate-induced inactivation by L-serine or by beta-chloro-L-alanine. The rates of inactivation (kinact) are similar to the rates of catalysis (kcat). The partition ratios are very low (kcat/kinact = 0.25-3) and are affected by alpha subunit ligands and monovalent cations. The inactivation product released by alkali was shown by HPLC and by fluorescence, absorption, and mass spectroscopy to be identical to a compound previously synthesized from pyridoxal phosphate and pyruvate. We suggest that alterations in the cofactor chemistry that result from the engineered Asp377 in the active site of the beta subunit may promote the mechanism-based inactivation.     

Altering substrate specificity at the heme edge of cytochrome c peroxidase

Two mutants of cytochrome c peroxidase (CCP) are reported which exhibit unique specificities toward oxidation of small substrates. Ala-147 in CCP is located near the delta-meso edge of the heme and along the solvent access channel through which H2O2 is thought to approach the active site. This residue was replaced with Met and Tyr to investigate the hypothesis that small molecule substrates are oxidized at the exposed delta-meso edge of the heme. X-ray crystallographic analyses confirm that the side chains of A147M and A147Y are positioned over the delta-meso heme position and might therefore modify small molecule access to the oxidized heme cofactor. Steady-state kinetic measurements show that cytochrome c oxidation is enhanced 3-fold for A147Y relative to wild type, while small molecule oxidation is altered to varying degrees depending on the substrate and mutant. For example, oxidation of phenols by A147Y is reduced to less than 20% relative to the wild-type enzyme, while Vmax/e for oxidation of other small molecules is less affected by either mutation. However, the "specificity" of aniline oxidation by A147M, i.e., (Vmax/e)/Km, is 43-fold higher than in wild-type enzyme, suggesting that a specific interaction for aniline has been introduced by the mutation. Stopped-flow kinetic data show that the restricted heme access in A147Y or A147M slows the reaction between the enzyme and H202, but not to an extent that it becomes rate limiting for the oxidation of the substrates examined. The rate constant for compound ES formation with A147Y is 2.5 times slower than wild-type CCP. These observations strongly support the suggestion that small molecule oxidations occur at sites on the enzyme distinct from those utilized by cytochrome c and that the specificity of small molecule oxidation can be significantly modulated by manipulating access to the heme edge. The results help to define the role of alternative electron transfer pathways in cytochrome c peroxidase and may have useful applications in improving the specificity of peroxidase with engineered function.