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B S Cooperman

Publications and source records attributed to B S Cooperman.

At least 19 recordsLinked to original sources
PubMed2001

Ligand binding sites in Escherichia coli inorganic pyrophosphatase: effects of active site mutations.

Type I soluble inorganic pyrophosphatases (PPases) are well characterized both structurally and mechanistically. Earlier we measured the effects of active site substitutions on pH--rate profiles for the type I PPases from both Escherichia coli (E-PPase) and Saccharomyces cerevisae (Y-PPase). Here we extend these studies by measuring the effects of such substitutions on the more discrete steps of ligand binding to E-PPase, including (a) Mg(2+) and Mn(2+) binding in the absence of added ligand; (b) Mg(2+) binding in the presence of either P(i) or hydroxymethylbisphosphonate (HMBP), a competitive inhibitor of E-PPase; and (c) P(i) binding in the presence of Mn(2+). The active site of a type I PPase has well-defined subsites for the binding of four divalent metal ions (M1--M4) and two phosphates (P1, P2). Our results, considered in light of pertinent results from crystallographic studies on both E-PPase and Y-PPase and parallel functional studies on Y-PPase, allow us to conclude the following: (a) residues E20, D65, D70, and K142 play key roles in the functional organization of the active site; (b) the major structural differences between the product and substrate complexes of E-PPase are concentrated in the lower half of the active site; (c) the M1 subsite is functionally isolated from the rest of the active site; and (d) the M4 subsite is an especially unconstrained part of the active site.

Amino Acid Substitution↗
PubMed2001

Toward a quantum-mechanical description of metal-assisted phosphoryl transfer in pyrophosphatase.

The wealth of kinetic and structural information makes inorganic pyrophosphatases (PPases) a good model system to study the details of enzymatic phosphoryl transfer. The enzyme accelerates metal-complexed phosphoryl transfer 10(10)-fold: but how? Our structures of the yeast PPase product complex at 1.15 A and fluoride-inhibited complex at 1.9 A visualize the active site in three different states: substrate-bound, immediate product bound, and relaxed product bound. These span the steps around chemical catalysis and provide strong evidence that a water molecule (O(nu)) directly attacks PPi with a pK(a) vastly lowered by coordination to two metal ions and D117. They also suggest that a low-barrier hydrogen bond (LBHB) forms between D117 and O(nu), in part because of steric crowding by W100 and N116. Direct visualization of the double bonds on the phosphates appears possible. The flexible side chains at the top of the active site absorb the motion involved in the reaction, which may help accelerate catalysis. Relaxation of the product allows a new nucleophile to be generated and creates symmetry in the elementary catalytic steps on the enzyme. We are thus moving closer to understanding phosphoryl transfer in PPases at the quantum mechanical level. Ultra-high resolution structures can thus tease out overlapping complexes and so are as relevant to discussion of enzyme mechanism as structures produced by time-resolved crystallography.

Crystallography, X-Ray↗
PubMed2001

A quantitative model for allosteric control of purine reduction by murine ribonucleotide reductase.

The reduction of purine nucleoside diphosphates by murine ribonucleotide reductase requires catalytic (R1) and free radical-containing (R2) enzyme subunits and deoxynucleoside triphosphate allosteric effectors. A quantitative 16 species model is presented, in which all pertinent equilibrium constants are evaluated, that accounts for the effects of the purine substrates ADP and GDP, the deoxynucleoside triphosphate allosteric effectors dGTP and dTTP, and the dimeric murine R2 subunit on both the quaternary structure of murine R1 subunit and the dependence of holoenzyme (R1(2)R2(2)) activity on substrate and effector concentrations. R1, monomeric in the absence of ligands, dimerizes in the presence of substrate, effectors, or R2(2) because each of these ligands binds R1(2) with higher affinity than R1 monomer. This leads to apparent positive heterotropic cooperativity between substrate and allosteric effector binding that is not observed when binding to the dimeric protein itself is evaluated. Allosteric activation results from an increase in k(cat) for substrate reduction upon binding of the correct effector, rather than from heterotropic cooperativity between effector and substrate. Neither the allosteric site nor the active site displays nucleotide base specificity: dissociation constants for dGTP and dTTP are nearly equivalent and K(m) and k(cat) values for both ADP and GDP are similar. R2(2) binding to R1(2) shows negative heterotropic cooperativity vis-à-vis effectors but positive heterotropic cooperativity vis-à-vis substrates. Binding of allosteric effectors to the holoenzyme shows homotropic cooperativity, suggestive of a conformational change induced by activator binding. This is consistent with kinetic results indicating full dimer activation upon binding a single equivalent of effector per R1(2)R2(2).

Allosteric Regulation↗
PubMed2001

Probing essential water in yeast pyrophosphatase by directed mutagenesis and fluoride inhibition measurements.

The pattern of yeast pyrophosphatase (Y-PPase) inhibition by fluoride suggests that it replaces active site Mg(2+)-bound nucleophilic water, for which two different locations were proposed previously. To localize the bound fluoride, we investigate here the effects of mutating Tyr(93) and five dicarboxylic amino acid residues forming two metal binding sites in Y-PPase on its inhibition by fluoride and its five catalytic functions (steady-state PP(i) hydrolysis and synthesis, formation of enzyme-bound PP(i) at equilibrium, phosphate-water oxygen exchange, and Mg(2+) binding). D117E substitution had the largest effect on fluoride binding and made the P-O bond cleavage step rate-limiting in the catalytic cycle, consistent with the mechanism in which the nucleophile is coordinated by two metal ions and Asp(117). The effects of the mutations on PP(i) hydrolysis (as characterized by the catalytic constant and the net rate constant for P-O bond cleavage) were in general larger than on PP(i) synthesis (as characterized by the net rate constant for PP(i) release from active site). The effects of fluoride on the Y-PPase variants confirmed that PPase catalysis involves two enzyme.PP(i) intermediates, which bind fluoride with greatly different rates (Baykov, A. A., Fabrichniy, I. P., Pohjanjoki, P., Zyryanov, A. B., and Lahti, R. (2000) Biochemistry 39, 11939-11947). A mechanism for the structural changes underlying the interconversion of the enzyme.PP(i) intermediates is proposed.

Amino Acid Substitution↗
PubMed2001

Toward a rational design of peptide inhibitors of ribonucleotide reductase: structure-function and modeling studies.

Mammalian ribonucleotide reductase, a chemotherapeutic target, has two subunits, mR1 and mR2, and is inhibited by AcF(1)TLDADF(7), denoted P7. P7 corresponds to the C-terminus of mR2 and competes with mR2 for binding to mR1. We report results of a structure-function analysis of P7, obtained using a new assay measuring peptide ligand binding to mR1, that demonstrate stringent specificity for Phe at F(7), high specificity for Phe at F(1), and little specificity for the N-acyl group. They support a structural model in which the dominant interactions of P7 occur at two mR1 sites, the F(1) and F(7) subsites. The model is constructed from the structure of Escherichia coli R1 (eR1) complexed with the C-terminal peptide from eR2, aligned sequences of mR1 and eR1, and the trNOE-derived structure of mR1-bound P7. Comparison of this model with similar models constructed for mR1 complexed with other inhibitory ligands indicates that increased F(1) subsite interaction can offset lower F(7) subsite interaction and suggests strategies for the design of new, higher affinity inhibitors.

Animals↗
PubMed2001

Sequencing cyclic peptide inhibitors of mammalian ribonucleotide reductase by electrospray ionization mass spectrometry.

Mammalian ribonucleotide reductase (mRR) is a potential target for cancer intervention. A series of lactam-bridged cyclic peptide inhibitors (1-9) of mRR have been synthesized and tested in previous work. These inhibitors consist of cyclic and linear regions, causing their mass spectral characterization to be a challenge. We determined the fragmentation mechanism of cyclic peptides 1-9 using an ion-trap mass spectrometer equipped with an ESI source. Low-energy collision-induced dissociation of sodiated cyclic peptides containing linear branches follows a general pathway. Fragmentation of the linear peptide region produced mainly a and b ions. The ring peptide region was more stable and ring opening required higher collision energy, mainly occurring at the amide bond adjacent to the lactam bridge. The sodium ion, which bound to the carbonyl oxygen of the lactam bridge, acted as a fixed charge site and directed a charge-remote, sequence-specific fragmentation of the ring-opened peptide. Amino acid residues were cleaved sequentially from the C-terminus to the N-terminus. Our findings have established a new way to sequence cyclic peptides containing a lactam bridge based on charge-remote fragmentation. This methodology will permit unambiguous identification of high-affinity ligands within cyclic peptide libraries.

Enzyme Inhibitors↗
PubMed2000

Catalytically important ionizations along the reaction pathway of yeast pyrophosphatase.

Five catalytic functions of yeast inorganic pyrophosphatase were measured over wide pH ranges: steady-state PP(i) hydrolysis (pH 4. 8-10) and synthesis (6.3-9.3), phosphate-water oxygen exchange (pH 4. 8-9.3), equilibrium formation of enzyme-bound PP(i) (pH 4.8-9.3), and Mg(2+) binding (pH 5.5-9.3). These data confirmed that enzyme-PP(i) intermediate undergoes isomerization in the reaction cycle and allowed estimation of the microscopic rate constant for chemical bond breakage and the macroscopic rate constant for PP(i) release. The isomerization was found to decrease the pK(a) of the essential group in the enzyme-PP(i) intermediate, presumably nucleophilic water, from >7 to 5.85. Protonation of the isomerized enzyme-PP(i) intermediate decelerates PP(i) hydrolysis but accelerates PP(i) release by affecting the back isomerization. The binding of two Mg(2+) ions to free enzyme requires about five basic groups with a mean pK(a) of 6.3. An acidic group with a pK(a) approximately 9 is modulatory in PP(i) hydrolysis and metal ion binding, suggesting that this group maintains overall enzyme structure rather than being directly involved in catalysis.

Binding Sites↗
PubMed2000

Structure-based optimization of peptide inhibitors of mammalian ribonucleotide reductase.

Mammalian ribonucleotide reductase (mRR), a potential target for cancer intervention, is composed of two subunits, mR1 and mR2, whose association is critical for enzyme activity. In this article we describe the structural features of the mRR-inhibitor Ac-F-c[ELAK]-DF (Peptide 3) while bound to the mR1 subunit as determined by transferred NOEs. Peptide 3 is a cyclic analogue of the N-acetylated form of the heptapeptide C-terminus of the mR2 subunit (Ac-FTLDADF), which is the link between the two subunits and previously shown to be the minimal sequence inhibitor mRR by competing with mR2 for binding to mR1. Structural refinement employing an ensemble-based, full-relaxation matrix approach resulted in two structures varying in the conformations of F(1) and the cyclic lactam side chains of E(2) and K(5). The remainder of the molecule, both backbone and side chains, is extremely well-defined, with an RMSD of 0.54 A. The structural features of this conformationally constrained analogue provide unique insight into the requirements for binding to mR1, critical for further inhibitor development.

Animals↗
PubMed2000

Formation of the covalent chymotrypsin.antichymotrypsin complex involves no large-scale movement of the enzyme.

alpha(1)-Antichymotrypsin is a member of the serine proteinase inhibitor, or serpin, family that typically forms very long-lived, enzymatically inactive 1:1 complexes (denoted E*I*) with its target proteinases. Serpins share a conserved tertiary structure, in which an exposed region of amino acid residues (called the reactive center loop or RCL) acts as bait for a target proteinase. Within E*I*, the two proteins are linked covalently as a result of nucleophilic attack by Ser(195) of the serine proteinase on the P1 residue within the RCL of the serpin. This species is formally similar to the acyl enzyme species normally seen as an intermediate in serpin proteinase catalysis. However, its subsequent hydrolysis is extremely slow as a result of structural changes within the enzyme leading to distortion of the active site. There is at present an ongoing debate concerning the structure of the E*I* complex; in particular, as to whether the enzyme, bound to P1, maintains its original position at the top of the serpin molecule or instead translocates across the entire length of the serpin, with concomitant insertion of RCL residues P1-P14 within beta-sheet A and a large separation of the enzyme and RCL residue P1'. We report time-resolved fluorescence energy transfer and rapid mixing/quench studies that support the former model. Our results indicate that the distance between residue P1' in alpha(1)-antichymotrypsin and the amino terminus of chymotrypsin actually decreases on conversion of the encounter complex E.I to E*I*. These results led us to formulate a comprehensive mechanism that accounted both for our results and for those of others supporting the two different E*I* structures. In this mechanism, partial insertion of the RCL, with no large perturbation of the P1' enzyme distance, is followed by covalent acyl enzyme formation. Full insertion can subsequently take place, in a reversible fashion, with the position of equilibrium between the partially and fully inserted complexes depending on the particular serpin-proteinase pair under consideration.

Animals↗
PubMed2000

Ribosomal protein L2 is involved in the association of the ribosomal subunits, tRNA binding to A and P sites and peptidyl transfer.

Ribosomal proteins L2, L3 and L4, together with the 23S RNA, are the main candidates for catalyzing peptide bond formation on the 50S subunit. That L2 is evolutionarily highly conserved led us to perform a thorough functional analysis with reconstituted 50S particles either lacking L2 or harboring a mutated L2. L2 does not play a dominant role in the assembly of the 50S subunit or in the fixation of the 3'-ends of the tRNAs at the peptidyl-transferase center. However, it is absolutely required for the association of 30S and 50S subunits and is strongly involved in tRNA binding to both A and P sites, possibly at the elbow region of the tRNAs. Furthermore, while the conserved histidyl residue 229 is extremely important for peptidyl-transferase activity, it is apparently not involved in other measured functions. None of the other mutagenized amino acids (H14, D83, S177, D228, H231) showed this strong and exclusive participation in peptide bond formation. These results are used to examine critically the proposed direct involvement of His229 in catalysis of peptide synthesis.

Amino Acid Sequence↗
PubMed2000

The preparation and catalytic properties of recombinant human prostate-specific antigen (rPSA).

The serine proteinase prostate-specific antigen (PSA), and its complex with the serine proteinase inhibitor alpha(1)-antichymotrypsin (ACT), have been used as markers for the diagnosis of prostate cancer. PSA prepared from seminal fluid is typically contaminated with the trypsin-like glandular kallikrein (hK2). Here we describe a convenient and reproducible preparation of catalytically active recombinant PSA (rPSA) and demonstrate an overall similarity in the properties of cloned and refolded rPSA to PSA purified from seminal fluid. We also present results that are relevant for increasing the sensitivity of assays of PSA activity in biological fluids, for the putative role of PSA activity in physiologically important processes, including prostate cancer metastasis, and for the design of PSA inhibitors. Specifically, we find that added salts, in particular NaCl, give rise to dramatic increases in rPSA catalytic activity, as does added glycerol. On the other hand, Zn(2+), spermine, and spermidine, each a major component of seminal and prostatic fluid, strongly inhibit rPSA activity, with Zn(2+) being a non-competitive inhibitor while spermine is a competitive inhibitor. Citrate, also a major component of seminal and prostatic fluid, spermine, and spermidine each protect rPSA from Zn(2+) inhibition, presumably via Zn(2+) sequestration. Finally, rPSA efficiently proteolyzes several protein substrates.

Caseins↗
PubMed2000

Identification of 50S components neighboring 23S rRNA nucleotides A2448 and U2604 within the peptidyl transferase center of Escherichia coli ribosomes.

The 23S rRNA nucleotides 2604-12 and 2448-58 fall within the central loop of domain V, which forms a major part of the peptidyl transferase center of the ribosome. We report the synthesis of radioactive, photolabile 2'-O-methyloligoRNAs, PHONTs 1 and 2, complementary to these nucleotides and their exploitation in identifying 50S ribosomal subunit components neighboring their target sites. Photolysis of the 50S complex with PHONT 1, complementary to nts 2604-12, leads to target site-specific photoincorporation into protein L2 and 23S rRNA nucleotides A886, Alpha1918, A1919, G1922-C1924, U2563, U2586, and C2601. Photolysis of the 50S complex with PHONT 2, complementary to nts 2448-58, leads to target site-specific probe photoincorporation into proteins L2, L3, one or more of proteins L17, L18, L21, and of proteins L9, L15, L16, and 23S rRNA nucleotides C2456 and psi2457. Chemical footprinting studies show that 2'-O-methyloligoRNA binding causes little distortion of the peptidyl transferase center but do provide suggestive evidence for the location of flexible regions within 23S rRNA. The significance of these results for the structure of the peptidyl transferase center is considered.

Adenine Nucleotides↗
PubMed1999

Identification of 23S rRNA nucleotides neighboring the P-loop in the Escherichia coli 50S subunit.

We report the synthesis of a radioactive, photolabile 2'-O-methyloligoRNA probe, 2258-53/52(SAz)-48, PHONT1, and its exploitation in identifying 23S rRNA nucleotides neighboring the so-called 'P-loop'. The probe is complementary to nt 2248-2258 in Escherichia coli 50S subunits. PHONT1 contains a p-azidophenacyl group attached to a phosphorothioate bridge between the nucleotides complementary to the positions 2252-2253, such that the photogenerated nitrene is maximally 17-19 A from 23S RNA nucleotides G2252 and G2253. PHONT1 binds to the 50S subunit, and photoincorporates within or immediately adjacent to its target site, as well as into several nucleotides falling between G2357 and A2430. The significance of these results for the structure of the peptidyl transferase center is considered. The PHONT approach is generally applicable to studies of complex RNA-containing molecules.

Azides↗
PubMed1999

Synthesis and biological activity of cyclic peptide inhibitors of ribonucleotide reductase.

[formula: see text] A series of lactam-bridged peptide inhibitors (2-6) of mammalian ribonucleotide reductase (mRR) has been designed and synthesized on the basis of the heptapeptide N-AcFTLDADF (1), corresponding to the C-terminus of the R2 subunit of mRR. Inhibition studies revealed a direct relation between ring size and activity, with peptide 5 being 2.5 times more potent than peptide 1.

Enzyme Inhibitors↗
PubMed1999

Evolutionary aspects of inorganic pyrophosphatase.

Based on the primary structure, soluble inorganic pyrophosphatases can be divided into two families which exhibit no sequence similarity to each other. Family I, comprising most of the known pyrophosphatase sequences, can be further divided into prokaryotic, plant and animal/fungal pyrophosphatases. Interestingly, plant pyrophosphatases bear a closer similarity to prokaryotic than to animal/fungal pyrophosphatases. Only 17 residues are conserved in all 37 pyrophosphatases of family I and remarkably, 15 of these residues are located at the active site. Subunit interface residues are conserved in animal/fungal but not in prokaryotic pyrophosphatases.

Amino Acid Sequence↗
PubMed1999

Antichymotrypsin interaction with chymotrypsin. Intermediates on the way to inhibited complex formation.

Serpins form enzymatically inactive covalent complexes (designated E*I*) with their target proteinases, corresponding most likely to the acyl enzyme that resembles the normal intermediate in substrate turnover. Formation of E*I* involves large changes in the conformation of the reactive center loop (residues P17 to P9') and of the serpin molecule in general. The "hinge" region of the reactive center loop, including residues P10-P14, shows facile movement in and out of beta-sheet A, and this movement appears to be crucial in determining whether E*I* is formed (the inhibitor pathway) or whether I is rapidly hydrolyzed to I* (the substrate pathway). Here, we report stopped-flow and rapid quench studies investigating the pH dependence of the conversion of the alpha1-antichymotrypsin.alpha-chymotrypsin encounter complex, E.I, to E*I*. These studies utilize fluorescent derivatives of cysteine variants of alpha1-antichymotrypsin at the P11 and P13 residues. Our results demonstrate three identifiable intermediates, EIa, EIb, and EIc, between E.I and E*I* and permit informed speculation regarding the nature of these intermediates. Partitioning between inhibitor and substrate pathways occurs late in the process of E*I* formation, most likely from a species occurring between EIc and E*I*.

Animals↗