Dispensability of glutamic acid 48 and aspartic acid 134 for Mn2+-dependent activity of Escherichia coli ribonuclease HI

The activities of the eight mutant proteins of Escherichia coli RNase HI, in which the four carboxylic amino acids (Asp(10), Glu(48), Asp(70), and Asp(134)) involved in catalysis are changed to Asn (Gln) or Ala, were examined in the presence of Mn(2+). Of these proteins, the E48A, E48Q, D134A, and D134N proteins exhibited the activity, indicating that Glu(48) and Asp(134) are dispensable for Mn(2+)-dependent activity. The maximal activities of the E48A and D134A proteins were comparable to that of the wild-type protein. However, unlike the wild-type protein, these mutant proteins exhibited the maximal activities in the presence of >100 microM MnCl(2), and their activities were not inhibited at higher Mn(2+) concentrations (up to 10 mM). The wild-type protein contains two Mn(2+) binding sites and is activated upon binding of one Mn(2+) ion at site 1 at low ( approximately 1 microM) Mn(2+) concentrations. This activity is attenuated upon binding of a second Mn(2+) ion at site 2 at high (>10 microM) Mn(2+) concentrations. The cleavage specificities of the mutant proteins, which were examined using oligomeric substrates at high Mn(2+) concentrations, were identical to that of the wild-type protein at low Mn(2+) concentrations but were different from that of the wild-type protein at high Mn(2+) concentrations. These results suggest that one Mn(2+) ion binds to the E48A, E48Q, D134A, and D134N proteins at site 1 or a nearby site with weaker affinities. The binding analyses of the Mn(2+) ion to these proteins in the absence of the substrate support this hypothesis. When Mn(2+) ion is used as a metal cofactor, the Mn(2+) ion itself, instead of Glu(48) and Asp(134), probably holds water molecules required for activity.     

Intrinsic double-stranded-RNA processing activity of Escherichia coli ribonuclease III lacking the dsRNA-binding domain.

The ribonuclease III superfamily represents a structurally related group of double-strand (ds) specific endoribonucleases which play key roles in diverse prokaryotic and eukaryotic RNA maturation and degradation pathways. A dsRNA-binding domain (dsRBD) is a conserved feature of the superfamily and is important for substrate recognition. RNase III family members also exhibit a "catalytic" domain, in part defined by a set of highly conserved amino acids, of which at least one (a glutamic acid) is important for cleavage but not for substrate binding. However, it is not known whether the catalytic domain requires the dsRBD for activity. This report shows that a truncated form of Escherichia coli RNase III lacking the dsRBD (RNase III[DeltadsRBD]) can accurately cleave small processing substrates in vitro. Optimal activity of RNase III[DeltadsRBD] is observed at low salt concentrations (<60 mM Na(+)), either in the presence of Mg(2+) (>25 mM) or Mn(2+) ( approximately 5 mM). At 60 mM Na(+) and 5 mM Mn(2+) the catalytic efficiency of RNase III[DeltadsRBD] is similar to that of RNase III at physiological salt concentrations and Mg(2+). In the presence of Mg(2+) RNase III[DeltadsRBD] is less efficient than the wild-type enzyme, due to a higher K(m). Similar to RNase III, RNase III[DeltadsRBD] is inhibited by high concentrations of Mn(2+), which is due to metal ion occupancy of an inhibitory site on the enzyme. RNase III[DeltadsRBD] retains strict specificity for dsRNA, as indicated by its inability to cleave (rA)(25), (rU)(25), or (rC)(25). Moreover, dsDNA, ssDNA, or an RNA-DNA hybrid are not cleaved. Low (micromolar) concentrations of ethidium bromide block RNase III[DeltadsRBD] cleavage of substrate, which is similar to the inhibition seen with RNase III and is indicative of an intercalative mode of inhibition. Finally, RNase III[DeltadsRBD] is sensitive to specific Watson-Crick base-pair substitutions which also inhibit RNase III. These findings support an RNase III mechanism of action in which the catalytic domain (i) can function independently of the dsRBD, (ii) is dsRNA-specific, and (iii) participates in cleavage site selection.     

Mutational analysis of the nuclease domain of Escherichia coli ribonuclease III. Identification of conserved acidic residues that are important for catalytic function in vitro

The ribonuclease III superfamily represents a structurally distinct group of double-strand-specific endonucleases with essential roles in RNA maturation, RNA decay, and gene silencing. Bacterial RNase III orthologs exhibit the simplest structures, with an N-terminal nuclease domain and a C-terminal double-stranded RNA-binding domain (dsRBD), and are active as homodimers. The nuclease domain contains conserved acidic amino acids, which in Escherichia coli RNase III are E38, E41, D45, E65, E100, D114, and E117. On the basis of a previously reported crystal structure of the nuclease domain of Aquifex aeolicus RNase III, the E41, D114, and E117 side chains of E. coli RNase III are expected to be coordinated to a divalent metal ion (Mg(2+) or Mn(2+)). It is shown here that the RNase III[E41A] and RNase III[D114A] mutants exhibit catalytic activities in vitro in 10 mM Mg(2+) buffer that are comparable to that of the wild-type enzyme. However, at 1 mM Mg(2+), the activities are significantly lower, which suggests a weakened affinity for metal. While RNase III[E41A] and RNase III[D114A] have K(Mg) values that are approximately 2.8-fold larger than the K(Mg) of RNase III (0.46 mM), the RNase III[E41A/D114A] double mutant has a K(Mg) of 39 mM, suggesting a redundant function for the two side chains. RNase III[E38A], RNase III[E65A], and RNase III[E100A] also require higher Mg(2+) concentrations for optimal activity, with RNase III[E100A] exhibiting the largest K(Mg). RNase III[D45A], RNase III[D45E], and RNase III[D45N] exhibit negligible activities, regardless of the Mg(2+) concentration, indicating a stringent functional requirement for an aspartate side chain. RNase III[D45E] activity is partially rescued by Mn(2+). The potential functions of the conserved acidic residues are discussed in the context of the crystallographic data and proposed catalytic mechanisms.     

Heterodimer-based analysis of subunit and domain contributions to double-stranded RNA processing by Escherichia coli RNase III in vitro

Members of the RNase III family are the primary cellular agents of dsRNA (double-stranded RNA) processing. Bacterial RNases III function as homodimers and contain two dsRBDs (dsRNA-binding domains) and two catalytic sites. The potential for functional cross-talk between the catalytic sites and the requirement for both dsRBDs for processing activity are not known. It is shown that an Escherichia coli RNase III heterodimer that contains a single functional wt (wild-type) catalytic site and an inactive catalytic site (RNase III[E117A/wt]) cleaves a substrate with a single scissile bond with a k(cat) value that is one-half that of wt RNase III, but exhibits an unaltered K(m). Moreover, RNase III[E117A/wt] cleavage of a substrate containing two scissile bonds generates singly cleaved intermediates that are only slowly cleaved at the remaining phosphodiester linkage, and in a manner that is sensitive to excess unlabelled substrate. These results demonstrate the equal probability, during a single binding event, of placement of a scissile bond in a functional or nonfunctional catalytic site of the heterodimer and reveal a requirement for substrate dissociation and rebinding for cleavage of both phosphodiester linkages by the mutant heterodimer. The rate of phosphodiester hydrolysis by RNase III[E117A/wt] has the same dependence on Mg(2+) ion concentration as that of the wt enzyme, and exhibits a Hill coefficient (h) of 2.0+/-0.1, indicating that the metal ion dependence essentially reflects a single catalytic site that employs a two-Mg(2+)-ion mechanism. Whereas an E. coli RNase III mutant that lacks both dsRBDs is inactive, a heterodimer that contains a single dsRBD exhibits significant catalytic activity. These findings support a reaction pathway involving the largely independent action of the dsRBDs and the catalytic sites in substrate recognition and cleavage respectively.     

Strong nucleic acid binding to the Escherichia coli RNase HI mutant with two arginine residues at the active site

The biochemical properties of the mutant protein D10R/E48R of Escherichia coli RNase HI, in which Asp(10) and Glu(48) are both replaced by Arg, were characterized. This mutant protein has been reported to have metal-independent RNase H activity at acidic pH [Casareno et al. (1995) J. Am. Chem. Soc. 117, 11011-11012]. The far- and near-UV CD spectra of this mutant protein were similar to those of the wild-type protein, suggesting that the protein conformation is not markedly changed by these mutations. Nevertheless, we found that this mutant protein did not show any RNase H activity in vitro. Instead, it showed high-nucleic-acid-binding affinity. Protein footprinting analyses suggest that DNA/RNA hybrid binds to or around the presumed substrate-binding site of the protein. In addition, this mutant protein did not complement the temperature-sensitive growth phenotype of the rnhA mutant strain, E. coli MIC3001, even at pH 6.0, suggesting that it does not show RNase H activity in vivo as well. These results are consistent with a current model for the catalytic mechanism of the enzyme, in which Glu(48) is not responsible for Mg(2+) binding but is involved in the catalytic function.     

Mechanisms of activation of phosphoenolpyruvate carboxykinase from Escherichia coli by Ca2+ and of desensitization by trypsin

The 1.8-A resolution structure of the ATP-Mg(2+)-Ca(2+)-pyruvate quinary complex of Escherichia coli phosphoenolpyruvate carboxykinase (PCK) is isomorphous to the published complex ATP-Mg(2+)-Mn(2+)-pyruvate-PCK, except for the Ca(2+) and Mn(2+) binding sites. Ca(2+) was formerly implicated as a possible allosteric regulator of PCK, binding at the active site and at a surface activating site (Glu508 and Glu511). This report found that Ca(2+) bound only at the active site, indicating that there is likely no surface allosteric site. (45)Ca(2+) bound to PCK with a K(d) of 85 micro M and n of 0.92. Glu508Gln Glu511Gln mutant PCK had normal activation by Ca(2+). Separate roles of Mg(2+), which binds the nucleotide, and Ca(2+), which bridges the nucleotide and the anionic substrate, are implied, and the catalytic mechanism of PCK is better explained by studies of the Ca(2+)-bound structure. Partial trypsin digestion abolishes Ca(2+) activation (desensitizes PCK). N-terminal sequencing identified sensitive sites, i.e., Arg2 and Arg396. Arg2Ser, Arg396Ser, and Arg2Ser Arg396Ser (double mutant) PCKs altered the kinetics of desensitization. C-terminal residues 397 to 540 were removed by trypsin when wild-type PCK was completely desensitized. Phe409 and Phe413 interact with residues in the Ca(2+) binding site, probably stabilizing the C terminus. Phe409Ala, DeltaPhe409, Phe413Ala, Delta397-521 (deletion of residues 397 to 521), Arg396(TAA) (stop codon), and Asp269Glu (Ca(2+) site) mutations failed to desensitize PCK and, with the exception of Phe409Ala, appeared to have defects in the synthesis or assembly of PCK, suggesting that the structure of the C-terminal domain is important in these processes.     

Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis. 3. Changing metal specificity and the pH profile by site-directed mutagenesis.

Aldose-ketose isomerization by xylose isomerase requires bivalent cations such as Mg2+, Mn2+, or Co2+. The active site of the enzyme from Actinoplanes missouriensis contains two metal ions that are involved in substrate binding and in catalyzing a hydride shift between the C1 and C2 substrate atoms. Glu 186 is a conserved residue located near the active site but not in contact with the substrate and not with a metal ligand. The E186D and E186Q mutant enzymes were prepared. Both are active, and their metal specificity is different from that of the wild type. The E186Q enzyme is most active with Mn2+ and has a drastically shifted pH optimum. The X-ray analysis of E186Q was performed in the presence of xylose and either Mn2+ or Mg2+. The Mn2+ structure is essentially identical to that of the wild type. In the presence of Mg2+, the carboxylate group of residue Asp 255, which is part of metal site 2 and a metal ligand, turns toward Gln 186 and hydrogen bonds to its side-chain amide. Mg2+ is not bound at metal site 2, explaining the low activity of the mutant with this cation. Movements of Asp 255 also occur in the wild-type enzyme. We propose that they play a role in the O1 to O2 proton relay accompanying the hydride shift.     

Identification of single Mn(2+) binding sites required for activation of the mutant proteins of E.coli RNase HI at Glu48 and/or Asp134 by X-ray crystallography

Escherichia coli RNase HI has two Mn(2+)-binding sites. Site 1 is formed by Asp10, Glu48, and Asp70, and site 2 is formed by Asp10 and Asp134. Site 1 and site 2 have been proposed to be an activation site and an attenuation site, respectively. However, Glu48 and Asp134 are dispensable for Mn(2+)-dependent activity. In order to identify the Mn(2+)-binding sites of the mutant proteins at Glu48 and/or Asp134, the crystal structures of the mutant proteins E48A-RNase HI*, D134A-RNase HI*, and E48A/D134N-RNase HI* in complex with Mn(2+) were determined. In E48A-RNase HI*, Glu48 and Lys87 are replaced by Ala. In D134A-RNase HI*, Asp134 and Lys87 are replaced by Ala. In E48A/D134N-RNase HI*, Glu48 and Lys87 are replaced by Ala and Asp134 is replaced by Asn. All crystals had two or four protein molecules per asymmetric unit and at least two of which had detectable manganese ions. These structures indicated that only one manganese ion binds to the various positions around the center of the active-site pocket. These positions are different from one another, but none of them is similar to site 1. The temperature factors of these manganese ions were considerably larger than those of the surrounding residues. These results suggest that the first manganese ion required for activation of the wild-type protein fluctuates among various positions around the center of the active-site pockets. We propose that this fluctuation is responsible for efficient hydrolysis of the substrates by the protein (metal fluctuation model). The binding position of the first manganese ion is probably forced to shift to site 1 or site 2 upon binding of the second manganese ion.     

Metal ion binding and enzymatic mechanism of Methanococcus jannaschii RNase HII

RNase H degrades the RNA moiety in DNA:RNA hybrid in a divalent metal ion dependent manner. It is essential to understand the role of metal ion in enzymatic mechanism. One of the key points in this study is how many metal ions are involved in the enzyme catalysis. Accordingly, either one-metal binding mechanism or two-metal binding mechanism is proposed. We have studied the thermodynamic properties of four metal ions (Mg(2+), Mn(2+), Ca(2+), and Ba(2+)) binding to Methanococcus jannaschii RNase HII using isothermal titration calorimetry. All of the four metal ions were found to bind Mj RNase HII with 1:1 stoichiometry in the absence of substrate. Together with enzymatic activity assay data, we propose that only one metal ion binding to the enzyme in catalytic process. We also studied the pH dependence of metal binding and enzyme activity and found that at pH 6.5, Mg(2+) did not bind to the enzyme without the substrate but still activated the enzyme to about 2% of its maximum activity (in 10 mM Mn(2+) at pH 8). This implies that the substrate may also be incorporated in metal ion binding and help to position the metal ion. To find which acidic residues correspond to metal ion binding, we also studied the binding thermodynamics and enzymatic activity assay of four mutants: D7N, E8Q, D112N, and D149N in the presence of Mn(2+). The thermodynamic parameters are least affected for the D149N mutant, which has a very low enzymatic activity. This indicates that Asp149 is essential for the enzymatic activity. On the basis of all these observations, we suggest a metal binding model in which D7, E8, and D112 bind the metal ion and D149 activates a water molecule to attack the P-O bond in the RNA chain of the substrate.     

Identification of Asp218 and Asp326 as the principal Mg2+ binding ligands of the homing endonuclease PI-SceI

The monomeric homing endonuclease PI-SceI harbors two catalytic centers which cooperate in the cleavage of the two strands of its extended recognition sequence. Structural and biochemical data suggest that catalytic center I contains Asp218, Asp229, and Lys403, while catalytic center II contains Asp326, Thr341, and Lys301. The analogy with I-CreI, for which the cocrystal structure with the DNA substrate has been determined, suggests that Asp218 and Asp229 in catalytic center I and Asp326 and Thr341 in catalytic center II serve as ligands for Mg(2+), the essential divalent metal ion cofactor which can be replaced by Mn(2+) in vitro. We have carried out a mutational analysis of these presumptive Mg(2+) ligands. The variants carrying an alanine or asparagine substitution bind DNA, but (with the exception of the D229N variant) are inactive in DNA cleavage in the presence of Mg(2+), demonstrating that these residues are important for cleavage. Our finding that the PI-SceI variants carrying single cysteine substitutions at these positions are inactive in the presence of the oxophilic Mg(2+) but active in the presence of the thiophilic Mn(2+) suggests that the amino acid residues at these positions are involved in cofactor binding. From the fact that in the presence of Mn(2+) the D218C and D326C variants are even more active than the wild-type enzyme, it is concluded that Asp218 and Asp326 are the principal Mg(2+) ligands of PI-SceI. On the basis of these findings and the available structural information, a model for the composition of the two Mg(2+) binding sites of PI-SceI is proposed.     

Ribonuclease III cleavage of a bacteriophage T7 processing signal. Divalent cation specificity, and specific anion effects.

Escherichia coli ribonuclease III, purified to homogeneity from an overexpressing bacterial strain, exhibits a high catalytic efficiency and thermostable processing activity in vitro. The RNase III-catalyzed cleavage of a 47 nucleotide substrate (R1.1 RNA), based on the bacteriophage T7 R1.1 processing signal, follows substrate saturation kinetics, with a Km of 0.26 microM, and kcat of 7.7 min.-1 (37 degrees C, in buffer containing 250 mM potassium glutamate and 10 mM MgCl2). Mn2+ and Co2+ can support the enzymatic cleavage of the R1.1 RNA canonical site, and both metal ions exhibit concentration dependences similar to that of Mg2+. Mn2+ and Co2+ in addition promote enzymatic cleavage of a secondary site in R1.1 RNA, which is proposed to result from the altered hydrolytic activity of the metalloenzyme (RNase III 'star' activity), exhibiting a broadened cleavage specificity. Neither Ca2+ nor Zn2+ support RNase III processing, and Zn2+ moreover inhibits the Mg(2+)-dependent enzymatic reaction without blocking substrate binding. RNase III does not require monovalent salt for processing activity; however, the in vitro reactivity pattern is influenced by the monovalent salt concentration, as well as type of anion. First, R1.1 RNA secondary site cleavage increases as the salt concentration is lowered, perhaps reflecting enhanced enzyme binding to substrate. Second, the substitution of glutamate anion for chloride anion extends the salt concentration range within which efficient processing occurs. Third, fluoride anion inhibits RNase III-catalyzed cleavage, by a mechanism which does not involve inhibition of substrate binding.     

Engineering PQQ glucose dehydrogenase with improved substrate specificity. Site-directed mutagenesis studies on the active center of PQQ glucose dehydrogenase

Site-directed mutagenesis was carried out on the active site of water-soluble PQQ glucose dehydrogenase (PQQGDH-B) to improve its substrate specificity. Amino acid substitution of His168 resulted in a drastic decrease in the enzyme's catalytic activity, consistent with its putative catalytic role. Substitutions were also carried out in neighboring residues, Lys166, Asp167, and Gln169, in an attempt to alter the enzyme's substrate binding site. Lys166 and Gln169 mutants showed only minor changes in substrate specificity profiles. In sharp contrast, mutants of Asp167 showed considerably altered specificity profiles. Of the numerous Asp167 mutants characterized, Asp167Glu showed the best substrate specificity profile, while retaining most of its catalytic activity for glucose and stability. We also investigated the cumulative effect of combining the Asp167Glu substitution with the previously reported Asn452Thr mutation. Interpretation of the effect of the replacement of Asp167 to Glu on the alteration of substrate specificity in relation with the predicted 3D model of PQQGDH-B is also discussed.     

G350 of Escherichia coli RNase P RNA contributes to Mg2+ binding near the active site of the enzyme

G350 of Escherichia coli RNase P RNA is a highly conserved residue among all bacteria and lies near the known magnesium binding site for the RNase P ribozyme, helix P4. Mutations at G350 have a dramatic effect on substrate cleavage activity for both RNA alone and holoenzyme; the G350C mutation has the most severe phenotype. The G350C mutation also inhibits growth of cells that express the mutant RNA in vivo under conditions of magnesium starvation. The results suggest that G350 contributes to Mg(2+) binding at helix P4 of RNase P RNA.     

Crystallographic studies on the role of the C-terminal segment of human angiogenin in defining enzymatic potency

Human angiogenin (Ang) is an RNase in the pancreatic RNase superfamily that induces angiogenesis. Its catalytic activity is comparatively weak, but nonetheless critical for biological activity. The crystal structure of Ang has shown that enzymatic potency is attenuated in part by the obstructive positioning of Gln117 within the B(1) pyrimidine binding pocket, and that the C-terminal segment of residues 117-123 must reorient for Ang to bind and cleave RNA. The native closed conformation appears to be stabilized by Gln117-Thr44 and Asp116-Ser118 hydrogen bonds, as well as hydrophobic packing of Ile119 and Phe120. Consistent with this view, Q117G, D116H, and I119A/F120A variants are 4-30-fold more active than Ang. Here we have determined crystal structures for these variants to examine the structural basis for the activity increases. In all three cases, the C-terminal segment remains obstructive, demonstrating that none of the residues that has been replaced is essential for maintaining the closed conformation. The Q117G structure shows no changes other than the loss of the side chain of residue 117, whereas those of D116H and I119A/F120A reveal C-terminal perturbations beyond the replacement site, suggesting that the native closed conformation has been destabilized. Thus, the interactions of Gln117 seem to be less important than those of residues 116, 119, and 120 for stabilization. In D116H, His116 does not replicate either of the hydrogen bonds of Asp116 with Ser118 and instead forms a water-mediated interaction with catalytic residue His114; residues 117-121 deviate significantly from their positions in Ang. In I119A/F120A, the segment of residues 117-123 has become highly mobile and all of the interactions thought to position Gln117 have been weakened or lost; the space occupied by Phe120 in Ang is partially filled by Arg101, which has moved several angstroms. A crystal structure was also determined for the deletion mutant des(121-123), which has 10-fold reduced activity toward large substrates. The structure is consistent with the earlier proposal that residues 121-123 form part of a peripheral substrate binding subsite, but also raises the possibility that changes in the position of another residue, Lys82, might be responsible for the decreased activity of this variant.     

Mutations in the beta-subunit Thr(159) and Glu(184) of the Rhodospirillum rubrum F(0)F(1) ATP synthase reveal differences in ligands for the coupled Mg(2+)- and decoupled Ca(2+)-dependent F(0)F(1) activities

In the crystal structure of the mitochondrial F(1)-ATPase, the beta-Thr(163) residue was identified as a ligand to Mg(2+) and the beta-Glu(188) as directly involved in catalysis. We replaced the equivalent beta-Thr(159) of the chromatophore F(0)F(1) ATP synthase of Rhodospirillum rubrum with Ser, Ala, or Val and the Glu(184) with Gln or Lys. The mutant beta subunits were isolated and tested for their capacity to assemble into a beta-less chromatophore F(0)F(1) and restore its lost activities. All of them were found to bind into the beta-less enzyme with the same efficiency as the wild type beta subunit, but only the beta-Thr(159) --> Ser mutant restored the activity of the assembled enzyme. These results indicate that both Thr(159) and Glu(184) are not required for assembly and that Glu(184) is indeed essential for all the membrane-bound chromatophore F(0)F(1) activities. A detailed comparison between the wild type and the beta-Thr(159) --> Ser mutant revealed a rather surprising difference. Although this mutant restored the wild type levels and all specific properties of this F(0)F(1) proton-coupled ATP synthesis as well as Mg- and Mn-dependent ATP hydrolysis, it did not restore at all the proton-decoupled CaATPase activity. This clear difference between the ligands for Mg(2+) and Mn(2+), where threonine can be replaced by serine, and Ca(2+), where only threonine is active, suggests that the beta-subunit catalytic site has different conformational states when occupied by Ca(2+) as compared with Mg(2+). These different states might result in different interactions between the beta and gamma subunits, which are involved in linking F(1) catalysis with F(0) proton-translocation and can thus explain the complete absence of Ca-dependent proton-coupled F(0)F(1) catalytic activity.     

Mutations in the ribonuclease H active site of HIV-RT reveal a role for this site in stabilizing enzyme-primer-template binding

The RNase H activity of HIV-RT is coordinated by a catalytic triad (E478, D443, D498) of acidic residues that bind divalent cations. We examined the effect of RNase H deficient E(478)-->Q and D(549)-->N mutations that do not alter polymerase activity on binding of enzyme to various nucleic acid substrates. Binding of the mutant and wild-type enzymes to various nucleic acid substrates was examined by determining dissociation rate constants (k(off)) by titrating both Mg(2+) and salt concentrations. In agreement with the unaltered polymerase activity of the mutant, the k(off) values for the wild-type and mutant enzymes were essentially identical using DNA-DNA templates in the presence of 6 mM Mg(2+). However, with lower concentrations of Mg(2+) and in the absence of Mg(2+), although both enzymes dissociated more rapidly, the mutant enzymes dissociated several-fold more slowly than the wild type. This was also observed on RNA-DNA templates. These results indicate that alterations in residues essential for Mg(2+) binding have a pronounced positive effect on enzyme-template stability and that the negative residues in the RNase H region of the enzyme have a negative influence on binding in the absence of Mg(2+). In this regard RT is similar to other nucleic acid cleaving enzymes that show enhanced binding upon mutation of active site residues.     

Mutagenic analysis of functional residues in putative substrate-binding site and acidic domains of vacuolar H+-pyrophosphatase

Vacuolar H(+)-translocating inorganic pyrophosphatase (V-PPase) uses PP(i) as an energy donor and requires free Mg(2+) for enzyme activity and stability. To determine the catalytic domain, we analyzed charged residues (Asp(253), Lys(261), Glu(263), Asp(279), Asp(283), Asp(287), Asp(723), Asp(727), and Asp(731)) in the putative PP(i)-binding site and two conserved acidic regions of mung bean V-PPase by site-directed mutagenesis and heterologous expression in yeast. Amino acid substitution of the residues with alanine and conservative residues resulted in a marked decrease in PP(i) hydrolysis activity and a complete loss of H(+) transport activity. The conformational change of V-PPase induced by the binding of the substrate was reflected in the susceptibility to trypsin. Wild-type V-PPase was completely digested by trypsin but not in the presence of Mg-PP(i), while two V-PPase mutants, K261A and E263A, became sensitive to trypsin even in the presence of the substrate. These results suggest that the second acidic region is also implicated in the substrate hydrolysis and that at least two residues, Lys(261) and Glu(263), are essential for the substrate-binding function. From the observation that the conservative mutants K261R and E263D showed partial activity of PP(i) hydrolysis but no proton pump activity, we estimated that two residues, Lys(261) and Glu(263), might be related to the energy conversion from PP(i) hydrolysis to H(+) transport. The importance of two residues, Asp(253) and Glu(263), in the Mg(2+)-binding function was also suggested from the trypsin susceptibility in the presence of Mg(2+). Furthermore, it was found that the two acidic regions include essential common motifs shared among the P-type ATPases.     

Characterization of the active site of ADP-ribosyl cyclase.

ADP-ribosyl cyclase synthesizes two Ca(2+) messengers by cyclizing NAD to produce cyclic ADP-ribose and exchanging nicotinic acid with the nicotinamide group of NADP to produce nicotinic acid adenine dinucleotide phosphate. Recombinant Aplysia cyclase was expressed in yeast and co-crystallized with a substrate, nicotinamide. x-ray crystallography showed that the nicotinamide was bound in a pocket formed in part by a conserved segment and was near the central cleft of the cyclase. Glu(98), Asn(107) and Trp(140) were within 3.5 A of the bound nicotinamide and appeared to coordinate it. Substituting Glu(98) with either Gln, Gly, Leu, or Asn reduced the cyclase activity by 16-222-fold, depending on the substitution. The mutant N107G exhibited only a 2-fold decrease in activity, while the activity of W140G was essentially eliminated. The base exchange activity of all mutants followed a similar pattern of reduction, suggesting that both reactions occur at the same active site. In addition to NAD, the wild-type cyclase also cyclizes nicotinamide guanine dinucleotide to cyclic GDP-ribose. All mutant enzymes had at least half of the GDP-ribosyl cyclase activity of the wild type, some even 2-3-fold higher, indicating that the three coordinating amino acids are responsible for positioning of the substrate but not absolutely critical for catalysis. To search for the catalytic residues, other amino acids in the binding pocket were mutagenized. E179G was totally devoid of GDP-ribosyl cyclase activity, and both its ADP-ribosyl cyclase and the base exchange activities were reduced by 10,000- and 18,000-fold, respectively. Substituting Glu(179) with either Asn, Leu, Asp, or Gln produced similar inactive enzymes, and so was the conversion of Trp(77) to Gly. However, both E179G and the double mutant E179G/W77G retained NAD-binding ability as shown by photoaffinity labeling with [(32)P]8-azido-NAD. These results indicate that both Glu(179) and Trp(77) are crucial for catalysis and that Glu(179) may indeed be the catalytic residue.