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B S Cooperman

Publications and source records attributed to B S Cooperman.

At least 55 records · Page 3Linked to original sources
PubMed1996

The structural basis for pyrophosphatase catalysis.

BACKGROUND: Soluble inorganic pyrophosphatase (PPase), an essential enzyme central to phosphorus metabolism, catalyzes the hydrolysis of the phosphoanhydride bond in inorganic pyrophosphate. Catalysis requires divalent metal ions which affect the apparent pKas of the essential general acid and base on the enzyme, and the pKa of the substrate. Three to five metal ions are required for maximal activity, depending on pH and enzyme source. A detailed understanding of catalysis would aid both in understanding the nature of biological mechanisms of phosphoryl transfer, and in understanding the role of divalent cations. Without a high-resolution complex structure such a model has previously been unobtainable. RESULTS: We report the first two high-resolution structures of yeast PPase, at 2.2 and 2.0 A resolution with R factors of around 17%. One structure contains the two activating metal ions; the other, the product (MnPi)2 as well. The latter structure shows an extensive network of hydrogen bond and metal ion interactions that account for virtually every lone pair on the product phosphates. It also contains a water molecule/hydroxide ion bridging two metal ions and, uniquely, a phosphate bound to four Mn2+ ions. CONCLUSIONS: Our structure-based model of the PPase mechanism posits that the nucleophile is the hydroxide ion mentioned above. This aspect of the mechanism is formally analogous to the "two-metal ion' mechanism of alkaline phosphatase, exonucleases and polymerases. A third metal ion coordinates another water molecule that is probably the required general acid. Extensive Lewis acid coordination and hydrogen bonds provide charge shielding of the electrophile and lower the pKa of the leaving group. This "three-metal ion' mechanism is in detail different from that of other phosphoryl transfer enzymes, presumably reflecting how ancient the reaction is.

Alkaline Phosphatase↗
PubMed1996

Structural change in alpha-chymotrypsin induced by complexation with alpha 1-antichymotrypsin as seen by enhanced sensitivity to proteolysis.

Both human neutrophil elastase (HNE) and free chymotrypsin (Chtr) proteolyze Chtr within the complex that Chtr forms with antichymotrypsin (ACT). As free Chtr is stable both to self-digestion and to digestion by HNE, these results are indicative of a stability and/or conformational change in Chtr that accompanies complex formation. As determined by both N-terminal sequence analysis and matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS), the major initial sites of HNE cleavage of complexed Chtr are between gamma-chain residues A158/S159 and V188/S189. Significantly, this latter site is at the base of the S1 site that recognizes the P1 position of the serpin. A slower cleavage in the beta-chain between T139/G140 is also found. In addition, rACT is cleaved between residues V22/D23. The gamma-chain of complexed Chtr is also cleaved by free Chtr, but at different sites: L162/L163 and W172/G173. beta-Chain cleavages were also found between residues Q81/K82 and F114/S115. Cleavages similar to those described above were also found when Chtr was complexed with the L358F-rACT variant, but not for Chtr complexed with either of the smaller inhibitors bovine pancreatic trypsin inhibitor or turkey ovomucoid third domain, nor for the covalent adduct of Chtr with N-p-tosylphenylalanyl chloromethyl ketone. We conclude that the structural change in Chtr making it a proteinase substrate is coupled with the large conformational change in ACT following complex formation. Complexed Chtr is much less reactive toward proteolytic digestion in the presence of high salt than in its absence, in accord with the high-salt induced release of active enzyme from the Chtr.rACT complex and the suggestion that electrostatic interactions mediate the coupling of structural change between rACT and Chtr within the Chtr.rACT complex. Potential physiological consequences of this work are explored.

Amino Acid Sequence↗
PubMed1996

A site-directed mutagenesis study of Saccharomyces cerevisiae pyrophosphatase. Functional conservation of the active site of soluble inorganic pyrophosphatases.

We report the expression and initial characterization of 19 active-site variants of Saccharomyces cerevisiae inorganic pyrophosphatase (PPase), including measurements of thermostability, oligomeric structure and specific activity at pH 7.2. 13 of the 19 conservative substitutions resulted in at least a fivefold decrease in activity, indicating that these residues are important for yeast PPase catalysis. The E58D, D117E, D120E and D152E variants had no activity under the conditions tested, suggesting that Glu58, Asp117, Asp120 and Asp152 may have crucial roles in catalysis. The effects of the mutations on catalytic activity were very similar to those observed with the corresponding variants of Escherichia coli PPase, proving conclusively that the active site and mechanism of soluble PPases are conserved. The D71E variant was more thermostable and the K56R, R78K, D115E and K154R variants were more thermolabile than the wild-type enzyme, whereas subunit:subunit interactions were somewhat weakened by the K56R, R78K, Y89F and K154R substitutions. These results suggest that Lys56, Asp71, Arg78, Tyr89, Asp115 and Lys154 are structurally important for yeast PPase.

Binding Sites↗
PubMed1996

Structure of Escherichia coli inorganic pyrophosphatase at 2.2 A resolution.

The refined crystal structures of hexameric soluble inorganic pyrophosphatase from E. coli (E-PPase) are reported to R factors of 18.7 and 18.3% at 2.15 and 2.2 A, respectively. The first contains one independent monomer; the other, two independent monomers, in an R32 unit cell. Because the E-PPase monomer is small with a large open active site, there are relatively few hydrophobic interactions that connect the active-site loops to the five-stranded twisted beta-barrel that is the hydrophobic core of the molecule. The active-site loops are, however, held in place by interactions between monomers around the threefold and twofold symmetry axes of the D(3) hexamer. Consequently, mutations of active-site residues (such as Glu20 and Lysl04) often affect protein stability and oligomeric structure. Conversely, mutations of residues in the interface between monomers (such as His136 and Hisl40) not only affect oligomeric structure but also affect active-site function. The effects of the H136Q and H140Q variants can be explained by the extended ionic interaction between H140, D143 and H136' of the neighbouring monomer. This interaction is further buttressed by an extensive hydrogen-bonding network that appears to explain why the E-PPase hexamer is so stable and also why the H136Q and H140Q variant proteins are less stable as hexamers.

Journal Article↗
PubMed1996

Catalysis by Escherichia coli inorganic pyrophosphatase: pH and Mg2+ dependence.

Steady-state rates of PPi hydrolysis by Escherichia coli inorganic pyrophosphatase (E-PPase) were measured as a function of magnesium pyrophosphatase (substrate) and free Mg2+ ion (activator) in the pH range 6.0-10.0. Computer fitting of hydrolysis data in combination with direct measures of Mg2+ binding to enzyme has resulted in a model that quantitatively accounts for our results. The major features of this model are the following: (a) E-PPase catalysis proceeds both with three and with four (and possibly with five) Mg2+ ions per active site; (b) catalysis requires both an essential base and an essential acid, and the pKas of these groups are modulated by the stoichiometry of bound Mg2+; and (c) the four-metal route predominates for concentrations of free Mg2+>0.2mM. The model straightforwardly accounts for the apparent linkage between increased pKa of an essential base and activity requirements for higher Mg2+ concentration observed for several active site variants. Microscopic rate constants for overall catalysis of PPi-Pi equilibration were determined at pH 6.5-9.3 by combined analysis of enzyme-bound PPi formation and rates of PPi hydrolysis, PPi synthesis, and Pi-H2O oxygen exchange. The catalytic activity of E-PPase at saturating substrate increases toward PPi hydrolysis and decreases toward PPi synthesis and Pi-H2O oxygen exchange with increasing pH. These changes are mainly due to an increased rate of dissociation of the second released Pi and a decreased rate of enzyme-bound PPi synthesis from enzyme-bound Pi, respectively, as the pH is raised .

Catalysis↗
PubMed1996

Effect of E20D substitution in the active site of Escherichia coli inorganic pyrophosphatase on its quaternary structure and catalytic properties.

Glutamic acid 20 is an evolutionarily conserved residue found within the active site of the inorganic pyrophosphatase of Escherichia coli (E-PPase). Here we determine the effect of E20D substitution on the quaternary structure and catalytic properties of E-PPase. In contrast to wild-type enzyme, which is hexameric under a variety of conditions, E20D-PPase can be dissociated by dilution into nearly inactive trimers, as shown by electrophoresis of cross-linked enzyme, analytical ultracentrifugation, and measurement of catalytic activity as a function of enzyme concentration. Hexamer stability is increased in the presence of both substrate and Mg2+, is maximal at pH 6.5, and falls off sharply as the pH is lowered or raised from this value. Measured at saturating substrate, 20 mM Mg2+ and pH 7.2, E20D substitution (a) decreases activity towards inorganic pyrophosphate (PPi) hydrolysis and oxygen exchange between water and inorganic phosphate (P1), (b) increases the rate of net PPi synthesis, and (c) decreases the amount of enzyme-bound PPi in equilibrium with Pi in solution. Measurements of PPi hydrolysis rate as a function of both Mg2+ concentration and pH for the E20D variant show that its decreased activity is largely accounted for on the basis of an increased pKa of the catalytically essential base at the active site, and the need for a Mg2+ stoichiometry of 5 in the enzyme-substrate complex, similar to what is seen for the D97E variant. By contrast, wild-type PPase catalysis over a wide range of Mg2+ concentration and pH is dominated by an enzyme-substrate complex having a total of four Mg2+ ions. These results are consistent with a supporting role for Glu20 in PPase catalysis and demostrate that even conservative mutation at the active site can perturb the quaternary structure of the enzyme.

Binding Sites↗
PubMed1996

Crystallographic identification of metal-binding sites in Escherichia coli inorganic pyrophosphatase.

We report refined crystal structures of the hexameric soluble inorganic pyrophosphatase from Escherichia coli (E-PPase) to R-factors of 18.3% and 17.1% at 2.2 and 2.3 angstroms, respectively. Both structures contain two independent monomers in the asymmetric unit of an R32 cell. The difference between the structures is that the latter contains 1.5 Mg2+ ions per monomer. One metal ion binds to the "tight" metal-binding site identified by equilibrium dialysis studies, and is coordinated to Asp65, Asp70, and Asp102. The other metal ion, shared between two monomers at a hitherto unidentified metal-binding site in the dyad interface between trimers, is coordinated through water molecules to Asp26s and Asn24s from two monomers. The hexamers with metal bound to them are more tightly associated than the ones without metal bound to them. Combined with our other mechanistic and structural data, the results suggest that, at high metal concentrations, E-PPase may bind at least 4.5 metals per monomer: two in the active site before binding substrate, two with substrate, and 0.5 in the dyad interface. Glu20 interacts via a water molecule with Asp70 and appears in the related yeast PPase structure (Heikinheimo, manuscript in preparation) to be involved in binding the second metal ion. Magnesium ion therefore stabilizes the hexamer form through both direct and indirect effects. The direct effect is by tighter association at the subunit interface; the indirect effect occurs because magnesium stabilizes the correct conformation of the loop between Glu20 and Ile32, a loop involved a trimer-trimmer interactions. Our results thus provide a structural explanation for the solution studies that show that the E20D variant is partially hexameric and that the hexamer form can be stabilized by binding magnesium ion.

Amino Acids↗
PubMed1996

An unusual route to thermostability disclosed by the comparison of Thermus thermophilus and Escherichia coli inorganic pyrophosphatases.

The structures of Escherichia coli soluble inorganic pyrophosphatase (E-PPase) and Thermus thermophilus soluble inorganic pyrophosphatase (T-PPase) have been compared to find the basis for the superior thermostability of T-PPase. Both enzymes are D3 hexamers and crystallize in the same space group with very similar cell dimensions. Two rather small changes occur in the T-PPase monomer: a systematic removal of Ser residues and insertion of Arg residues, but only in the C-terminal part of the protein, and more long-range ion pairs from the C-terminal helix to the rest of the molecule. Apart from the first five residues, the three-dimensional structures of E-PPase and T-PPase monomers are very similar. The one striking difference, however, is in the oligomeric interactions. In comparison with an E-PPase monomer, each T-PPase monomer is skewed by about 1 A in the xy plane, is 0.3 A closer to the center of the hexamer in the z direction, and is rotated by approximately 7 degrees about its center of gravity. Consequently, there are a number of additional hydrogen bond and ionic interactions, many of which form an interlocking network that covers all of the oligomeric surfaces. The change can also be seen in local distortions of three small loops involved in the oligomeric interfaces. The complex rigid-body motion has the effect that the hexamer is more tightly packed in T-PPase: the amount of surface area buried upon oligomerization increases by 16%. The change is sufficiently large to account for all of the increased thermostability of T-PPase over E-PPase and further supports the idea that bacterial PPases, most active as hexamers or tetramers, achieve a large measure of their stabilization through oligomerization. Rigid-body motions of entire monomers to produce tighter oligomers may be yet another way in which proteins can be made thermophilic.

Amino Acid Sequence↗
PubMed1996

Arginine substitutions in the hinge region of antichymotrypsin affect serpin beta-sheet rearrangement.

A hallmark of serpin function is the massive beta-sheet rearrangement involving the insertion of the cleaved reactive loop into beta-sheet A as strand s4A. This structural transition is required for inhibitory activity. Small hydrophobic residues at P14 and P12 positions of the reactive loop facilitate this transition, since these residues must pack in the hydrophobic core of the cleaved serpin. Despite the radical substitution of arginine at the P12 position, the crystal structure of cleaved A347R antichymotrypsin reveals full strand s4A insertion with normal beta-sheet A geometry; the R347 side chain is buried in the hydrophobic protein core. In contrast, the structure of cleaved P14 T345R antichymotrypsin reveals substantial yet incomplete strand s4A insertion, without burial of the R345 side chain.

Animals↗
PubMed1995

Dissociation of hexameric Escherichia coli inorganic pyrophosphatase into trimers on His-136-->Gln or His-140-->Gln substitution and its effect on enzyme catalytic properties.

Each of the five histidines in Escherichia coli inorganic pyrophosphatase (PPase) was replaced in turn by glutamine. Significant changes in protein structure and activity were observed in the H136Q and H140Q variants only. In contrast to wild-type PPase, which is hexameric, these variants can be dissociated into trimers by dilution, as shown by analytical ultracentrifugation and cross-linking. Mg2+ and substrate stabilize the hexameric forms of both variants. The hexameric H136Q- and H140Q-PPases have the same binding affinities for magnesium ion as wild-type, but their hydrolytic activities under optimal conditions are, respectively, 225 and 110% of wild-type PPase, and their synthetic activities, 340 and 140%. The increased activity of hexameric H136Q-PPase results from an increase in the rate constants governing most of the catalytic steps in both directions. Dissociation of the hexameric H136Q and H140Q variants into trimers does not affect the catalytic constants for PPi hydrolysis between pH 6 and 9 but drastically decreases their affinities for Mg2PPi and Mg2+. These results prove that His-136 and His-140 are key residues in the dimer interface and show that hexamer formation improves the substrate binding characteristics of the active site.

Amino Acid Sequence↗
PubMed1995

Identification of lysines within alpha 1-antichymotrypsin important for DNA binding. An unusual combination of DNA-binding elements.

The human serum serine protease inhibitor (serpin) alpha 1-antichymotrypsin (ACT) appears to be unique among serpins in its ability to bind to double-stranded DNA. Using site-directed mutagenesis and chemical modification, a tri-lysine sequence (residues 210-212) falling within a solvent exposed loop and the C-terminal peptide containing two lysines (residues 391 and 396) were shown to be important for DNA binding. Mutation of residues 210-212 from lysines to either glutamates or threonines abolished DNA binding. The Lys210-Thr211-212 and Thr210-Th4(211)-Lys212 variants displayed reduced affinity for DNA, especially at higher ionic strength. Limited acetylation of rACT with acetic anhydride led to loss of DNA binding and, conversely, DNA protected rACT from acetylation. A combination of CNBr digestion, peptide separation, and peptide sequencing identified Lys396, two residues from the C terminus, as the most reactive lysine in rACT. Acetylation of Lys396 is strongly decreased in the presence of DNA. The double mutant K391T/K396T-rACT had very little affinity for DNA. The epsilon-amines of lysines 210-212 are 8-15 A across a cleft from the epsilon-amines in Lys391 and Lys396, and together these two elements may form an unusual DNA binding domain. Attempts to isolate a DNA sequence to which ACT binds specifically have been unsuccessful to date, raising the possibility that nonspecific binding of ACT to DNA suffices to account for the ACT found in certain cell nuclei. ACT variants not binding to double-stranded DNA retain ACT protease inhibitory activity, a potentially important result for the use of ACT variants as therapeutic agents.

Acetylation↗
PubMed1995

Cold lability of the mutant forms of Escherichia coli inorganic pyrophosphatase.

The variants of Escherichia coli pyrophosphatase carrying the substitutions Glu20-->Asp, His136-->Gln or His140-->Gln are inactivated, in contrast to the wild-type enzyme, at temperatures below 25 degrees C: their activity measured at 25 degrees C decreases with decreasing the temperature of the stock enzyme solution. The inactivation is completely reversible and is explained by cold-induced dissociation of these hexameric enzymes into less active trimers.

Aspartic Acid↗
PubMed1995

Structure and function analysis of Escherichia coli inorganic pyrophosphatase: is a hydroxide ion the key to catalysis?

Using site-directed mutagenesis, we have completed replacing all 17 putative active site residues of Escherichia coli inorganic pyrophosphatase (PPase). We report here the production of 11 new variant proteins and their initial characterization, including thermostability, hydrophobicity, oligomeric structure, and specific activity at pH 8. Studies of the pH-rate profiles of 12 variants containing substitutions for potentially essential residues showed that the effect of the mutation was always to increase the pKa of a basic group essential for both substrate binding and catalysis by 1-3 pH units. The D70E variant had the lowest activity at all pHs; the K29R, R43K, and K142R variants also had low kcat/Km values. The principal effect seen in the other variant proteins was higher and sharper pH optima; their pH-independent kcat and kcat/Km values changed at most by a factor of 8. Our results suggest that the most likely candidate for the essential basic group affected by all mutations in the active site is a hydroxide ion stabilized by coordination to the essential Mg2+ ions. Analyzing our results using the structure recently obtained for E. coli PPase [Kankare et al. (1994) Protein Eng. 7, 823-830] led us to identify a group of residues, centered around Asp70 and including Tyr55, Asp65, Asp67, Asp102, and Lys104, that we believe binds the magnesium ions that are critical for the activity, possibly by stabilizing the essential hydroxide. Others, including Lys29, Arg43, and Lys142, are more spread out and more positively charged. They appear to be involved in binding substrate and product. Tyr55 is also a key part of the hydrophobic core of E. coli PPase; when it or residues that interact with it are conservatively mutated, there are changes in the overall structure of the enzyme as assayed by thermostability, hydrophobicity, or oligomeric structure.

Bacterial Proteins↗
PubMed1995

Effect of D97E substitution on the kinetic and thermodynamic properties of Escherichia coli inorganic pyrophosphatase.

Aspartic acid 97 in the inorganic pyrophosphatase of Escherichia coli (E-PPase) has been identified as an evolutionarily conserved residue forming part of the active site [Cooperman et al. (1992) Trends Biochem. Sci. 17, 262-266]. Here we determine the effect of D97E substitution on several kinetic and thermodynamic properties of E-PPase, including rate and equilibrium constants for enzyme-catalyzed PPi.Pi equilibration at pH 7.2 and 8.0, Mg2+ affinity in the presence and absence of substrate, and the Mg2+ and pH dependence of kcat and Km. We find the major effects of D97E substitution are to (a) decrease markedly the pH-independence rates of both PPi hydrolysis and, especially, PPi resynthesis on the enzyme, (b) selectively destabilize both the EMg4PPi complex and the transition state between this complex and the EMg2(MgPi)2 complex, (c) raise the pKa of the basic group "essential" for PPi hydrolysis and for productive PPi binding by 1.5 and > 2.2 log units, respectively, (d) distort a site to which Mg2+ binds in the absence of substrate such that occupancy of the site by Mg2+ no longer confers enzymatic activity, and (e) decrease the affinity of one of the two Mg2+ ions that binds to enzyme in the presence of substrate. That this multiplicity of effects arises from a single Asp to Glu substitution suggests, in the absence of any evidence for a generalized structural change, a tightly integrated active site in which the perturbation induced by conservative substitution at a single location can have widespread functional effects.

Bacterial Proteins↗
PubMed1995

Ribosomal components neighboring the 2475 loop in Escherichia coli 50S subunits.

We report the synthesis of a radioactive, photolabile oligodeoxyribonucleotide probe and its exploitation in identifying 50S ribosomal subunit components neighboring its target site in 23S rRNA. The probe is complementary to 23S rRNA nucleotides 2475-2483, a single-stranded sequence (the 2475 loop) near the peptidyltransferase center of Escherichia coli ribosomes. On photolysis in the presence of 50S subunits, it site-specifically incorporates into proteins L1, L13, L16, L32, and L33 and into 23S rRNA nucleotides G2470, A2471, and G2472. These results provide clear evidence that C2475 in 23S rRNA is within 21 A (the distance between C2475 and the photogenerated nitrene) of proteins L1, L13, L16, L32, and L33. The implications of these results for the evolving model of the internal structure of the 50S subunit are considered.

Affinity Labels↗
PubMed1995

Photolabile oligoDNA probes of internal Escherichia coli ribosomal structure.

We are examining the spatial arrangement of ribosomal components in the vicinity of rRNA sequences of particular significance for ribosome structure and function through the use of radioactive, photolabile derivatives of oligoDNAs complementary to such sequences. The oligoDNA probes bind to their target sequences in intact ribosomal subunits, and, on photolysis, incorporate into neighboring ribosomal components. Labeled ribosomal proteins are subsequently identified by SDS-PAGE, RP-HPLC and immunoprecipitation. Labeled rRNA is identified at the nucleotide level by RNase H cleavage and primer extension. The results obtained provide a description of the neighborhood surrounding a particular rRNA sequence and impose important constraints on the evolving three-dimensional models of both the 30S and 50S subunits, since they typically oblige several ribosomal components to fall within defined distances of the targeted rRNA. Our results will be discussed in terms of these models, emphasizing their consistency or inconsistency.

Base Sequence↗
PubMed1994

Ribosomal components neighboring the conserved 518-533 loop of 16S rRNA in 30S subunits.

We report the synthesis of a radioactive, photolabile oligodeoxyribonucleotide probe complementary to 16S rRNA nucleotides 518-526 and its exploitation in identifying 30S ribosomal subunit components neighboring its target site in 16S rRNA. Nucleotides 518-526 lie within an almost universally conserved single-stranded loop that has been linked to the decoding region of Escherichia coli ribosomes. On photolysis in the presence of activated 30S ribosomes, the probe site-specifically incorporates into proteins S3, S4, S7, and S12 (identified by SDS-PAGE, RP-HPLC, and immunological analysis); nucleotides C525, C526, and G527 adjacent to its target binding site; and the 3'-terminus of 16S rRNA. When the probe is photoincorporated into 30S subunits subjected to brief cold inactivation (SI subunits), S7 labeling is increased compared to activated subunit incorporation, while S3, S4, and S12 labeling is decreased, as is labeling to nucleotides C525, C526, and G527; labeling at the 16S rRNA 3'-terminus appears unchanged. Longer cold inactivation of the 30S subunits (LI subunits) leads to decreases in the labeling of all components. These results provide clear evidence that C526 lies within 24 A (the distance between C526 and the photogenerated nitrene) of proteins S3, S4, S7, and S12 and the 3'-terminus of 16S rRNA. The identity of the tryptic digestion patterns of S7 labeled with the probe complementary to 16S rRNA nucleotides 518-526 and with a probe complementary to nucleotides 1397-1405 [Muralikrishna, P., & Cooperman, B. S. (1994) Biochemistry 33, 1392-1398] also provides evidence for proximity between C526 and G1405. Our results support the conclusion of Dontsova et al. [Dontsova, O., et al. (1992) EMBO J. 11, 3105-3116] in placing the 530 loop in close proximity to the decoding center of the 30S subunit but are apparently inconsistent with some protein-protein distances determined by neutron diffraction [Capel, M. S., et al. (1988) J. Mol. Biol. 200, 65-87]. This inconsistency suggests that a multistate model of subunit conformation may be required to account for the totality of results pertaining to the internal structure of the 30S subunit.

Autoradiography↗