Nature's transitory covalent bond.
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Disulfides↗
Biomedical subjects
R T Raines
Publications and source records attributed to R T Raines.
Protein disulfide isomerase in spore germination and cell division.
Protein disulfide isomerase (PDI) is a protein of the endoplasmic reticulum (ER) that is essential for the unscrambling of nonnative disulfide bonds. Here, we have determined the importance of PDI to both spore germination and vegetative cell division. To vary the concentration of PDI in the ER, we used plasmids that direct the expression of rat PDI fused at its N terminus to either the alpha-factor pre-pro segment or the alpha-factor pre sequence, and fused at its C terminus to either the mammalian (KDEL) or the yeast (HDEL) ER retention signal. Classical yeast genetic (tetrad) analyses, and plasmid loss and plasmid shuffling experiments were used to evaluate the ability of these constructs to complement haploid Saccharomyces cerevisiae cells in which the endogenous PDI1 gene had been deleted. We find that basal levels of PDI in the ER are sufficient for vegetative growth. In contrast, high levels of PDI in the ER are required for efficient spore germination. Thus, catalysis of the unscrambling of nonnative disulfide bonds in cellular proteins is more important during spore germination than during vegetative cell division.
Animals↗
The CXXC motif: imperatives for the formation of native disulfide bonds in the cell.
The rapid formation of native disulfide bonds in cellular proteins is necessary for the efficient use of cellular resources. This process is catalyzed in vitro by protein disulfide isomerase (PDI), with the PDI1 gene being essential for the viability of Saccharomyces cerevisiae. PDI is a member of the thioredoxin (Trx) family of proteins, which have the active-site motif CXXC. PDI contains two Trx domains as well as two domains unrelated to the Trx family. We find that the gene encoding Escherichia coli Trx is unable to complement PDI1 null mutants of S.cerevisiae. Yet, Trx can replace PDI if it is mutated to have a CXXC motif with a disulfide bond of high reduction potential and a thiol group of low pKa. Thus, an enzymic thiolate is both necessary and sufficient for the formation of native disulfide bonds in the cell.
Base Sequence↗
Substrate binding and turnover by the highly specific I-PpoI endonuclease.
Intron-encoded endonucleases are distinguished by their ability to catalyze the cleavage of double-stranded DNA with high specificity. I-PpoI endonuclease, an intron-encoded endonuclease from the slime mold Physarum polycephalum, is a small enzyme (2 x 20 kDa) that catalyzes the cleavage of a large asymmetric DNA sequence (15 base pairs). Here, the interactions of I-PpoI with its substrate were examined during both binding (in the absence of Mg2+) and catalysis (in the presence of Mg2+). Using circular permutation assays, I-PpoI was shown to bend its substrate by 38 +/- 4 degrees upon binding. Two independent methods, gel mobility shift assays and fluorescence polarization assays, revealed that I-PpoI binds tightly to its substrate. Values of Kd range from 3.3 to 112 nM, increasing with increasing NaCl concentration. Similar salt effects on the values of Km were observed during steady-state catalysis. At low salt concentrations, the value of kcat/Km for the cleavage of an oligonucleotide duplex approaches 10(8) M-1 s-1. Although other divalent cations can replace Mg2+, catalysis by I-PpoI is most efficient in the presence of an oxophilic metal ion that prefers an octahedral geometry: Mg2+ > Mn2+ > Ca2+ = Co2+ > Ni2+ > Zn2+. Together, these results provide the first chemical insight into substrate binding and turnover by an intron-encoded endonuclease.
Base Sequence↗
Contribution of a tyrosine side chain to ribonuclease A catalysis and stability.
An intricate architecture of covalent bonds and noncovalent interactions appear to position the side chain of Lys 41 properly within the active site of bovine pancreatic ribonuclease A (RNase A). One of these interactions arises from Tyr 97, which is conserved in all 41 RNase A homologues of known sequence. Tyr 97 has a solvent-inaccessible side chain that donates a hydrogen bond to the main-chain oxygen of Lys 41. Here, the role of Tyr 97 was examined by replacing Tyr 97 with a phenylalanine, alanine, or glycine residue. All three mutant proteins have diminished catalytic activity, with the value of Kcat being perturbed more significantly than that of Km. The free energies with which Y97F, Y97A, and Y97G RNase A bind to the rate-limiting transition state during the cleavage of poly(cytidylic acid) are diminished by 0.74, 3.3, and 3.8 kcal/mol, respectively. These results show that even though Tyr 97 is remote from the active site, its side chain contributes to catalysis. The role of Tyr 97 in the thermal stability of RNase A is large. The conformational free energies of native Y97F, Y97A, and Y97G RNase A are decreased by 3.54, 12.0, and 11.7 kcal/mol, respectively. The unusually large decrease in stability caused by the Tyr-->Phe mutation could result from a decrease in the barrier to isomerization of the Lys 41-Pro 42 peptide bond.
Animals↗
Production of human pancreatic ribonuclease in Saccharomyces cerevisiae and Escherichia coli.
Human pancreatic ribonuclease (HP-RNase) has considerable promise as a therapeutic agent. Structure-function analyses of HP-RNase have been impeded by the difficulty of obtaining the enzyme from its host. Here, a gene encoding HP-RNase was designed, synthesized, and inserted into two expression vectors that then direct the production of HP-RNase in Saccharomyces cerevisiae (fused to either an unmodified or a modified a-factor pre-pro segment) or Escherichia coli (fused to the pelB signal sequence). HP-RNase produced in S. cerevisiae was secreted into the medium as an active enzyme, isolable at 0.1-0.2 mg/liter of culture. This isolate was heterogeneous due to extensive glycosylation and incomplete maturation of the pre-pro segment. HP-RNase produced in E. coli with the pET expression system was purified from the insoluble fraction of the cell lysate. Renaturation of the reduced and denatured protein produced active, homogeneous enzyme recoverable at 1 mg/liter of culture. The N terminus of the HP-RNase produced from the bacterial expression system was processed fully in vivo. The yeast system, combined with techniques that allow detection of picograms of ribonuclease activity, offers a sensitive probe for studies of post-translational modification and secretory targeting in eukaryotic cells. The bacterial system enables studies both to reveal new structure-function relationships in ribonucleases and to evaluate the use of HP-RNase as a cytotoxin that is tolerated by the human immune system.
Amino Acid Sequence↗
Immunosuppressive activity of bovine seminal ribonuclease and its mode of action.
Two preparations of dimeric BS RNase-native and recombinant proteins caused identical immunosuppressive effects on MLC-stimulated human lymphocytes. The monomers of RNase A and BS RNase were ten times less active. The inhibitory effect on MLC-stimmulation was followed by 90% inhibition of cell-mediated lympholysis (CML) caused by BS RNase (10 micrograms/ml). This effect indicated that BS RNase suppressed the recognition phase of the cytotoxic reaction, resulting in inhibition of generation of cytotoxic effector cells. BS RNase exerted a similar effect on generation of cytotoxic LAK cells. Cytotoxic activity of LAK cells or CTLs against K562 target cells was abrogated only when BS RNase was added at the beginning of the sensitizing phase, but the cytotoxicity of effector cells in the destruction phase was not influenced. The effect of RNase A on the generation of cytotoxic cells was much less pronounced. To get more information about the site of action, the effect of BS RNase on early lymphocyte stimulation by PHA was investigated by using fluorescein cell probes. BS RNase (100 micrograms/ml) prevented a shift in fluorescein emission occurring within one hour of activation using fluorescein diacetate as a marker for changes in the cytoplasmic matrix. On the contrary, it did not block the shift in fluorescence emission when tested with diphenylhexatrien as a marker for changes in membrane fluidity. Furthermore the effect of BS RNase on expression of membrane antigens expressed on activated human lymphocytes was estimated. BS RNase significantly inhibited the expression of CD25, CD38 and CD71 antigens on PHA-, Con A- and MLC-stimulated human T and B lymphocytes. No substantial change in expression of these antigens was observed on IL-2-stimulated cells, but DNA synthesis was totally abrogated. These results indicate that the mode of action of BS RNase on activated T and B lymphocytes is based mainly on the suppressed expression of receptors for interleukin-2-alpha-chain and transferrin.
ADP-ribosyl Cyclase↗
Mechanism of ribonuclease cytotoxicity.
Bovine seminal ribonuclease (BS-RNase), a dimeric homolog of bovine pancreatic ribonuclease A (RNase A), is toxic to mammalian cells. In contrast to dimeric BS-RNase, a monomeric BS-RNase and RNase A are not cytotoxic and are bound tightly by cytosolic ribonuclease inhibitor. To elucidate the mechanism of ribonuclease cytotoxicity, we constructed a series of hybrid and semisynthetic enzymes and examined their properties. In five hybrid enzymes, divergent residues in BS-RNase were replaced with the analogous residues of RNase A so as to diminish an interaction with a putative cellular receptor. In a semisynthetic enzyme, the disulfide bonds that cross-link the monomeric subunits of dimeric BS-RNase were replaced with thioether bonds, which can withstand the reducing environment of the cytosol. Each hybrid and semisynthetic enzyme had ribonucleolytic and cytotoxic activities comparable with those of wild-type BS-RNase. These results suggest that dimeric BS-RNase (pI = 10.3) enters cells by adsorptive rather than receptor-mediated endocytosis and then evades cytosolic ribonuclease inhibitor so as to degrade cellular RNA. This mechanism accounts for the need for a cytosolic ribonuclease inhibitor and for the cytotoxicity of other homologs of RNase A.
Amino Acid Sequence↗
The essential function of protein-disulfide isomerase is to unscramble non-native disulfide bonds.
Protein-disulfide isomerase (PDI) is an abundant protein of the endoplasmic reticulum that catalyzes dithiol oxidation and disulfide bond reduction and isomerization using the active site CGHC. Haploid pdi1 delta Saccharomyces cerevisiae are inviable, but can be complemented with either a wild-type rat PDI gene or a mutant gene coding for CGHS PDI (shufflease). In contrast, pdi1 delta yeast cannot be complemented with a gene coding for SGHC PDI. In vitro, shufflease is an efficient catalyst for the isomerization of existing disulfide bonds but not for dithiol oxidation or disulfide bond reduction. SGHC PDI catalyzes none of these processes. These results indicate that in vivo protein folding pathways contain intermediates with non-native disulfide bonds, and that the essential role of PDI is to unscramble these intermediates.
Amino Acid Sequence↗
A residue to residue hydrogen bond mediates the nucleotide specificity of ribonuclease A.
Bovine pancreatic ribonuclease A (RNase A) catalyzes the cleavage of the P-O5 bond of RNA after residues bound in the enzyme's B1 subsite. This subsite binds to cytidine 30-fold more tightly than to uridine and > 10(5)-fold more tightly than to adenine. Structural studies had suggested that the hydroxyl group of Thr45 can interact directly with the base of a bound nucleotide. In contrast, the carboxylate group of Asp83 cannot interact directly with bound substrate but can accept a hydrogen bond from the hydroxyl group of Thr45. To assess the role of the Thr45-Asp83 hydrogen bond in catalysis, T45G, D83A and T45G/D83A RNase A were prepared and their abilities to catalyze the cleavage of various substrates were determined. The results indicate that the side-chain of Asp83 enhances catalysis of reactions in which uridine is bound in the B1 subsite, but that this enhancement relies on the side-chain of Thr45. In contrast, the side-chain of Asp83 does not contribute to catalysis of reactions with cytidine in the B1 subsite. Thermodynamic cycles derived from kinetic parameters for the cleavage of poly(U) indicate that the Thr45-Asp83 interaction contributes 1.2 kcal/mol to transition state stabilization, which is 0.9 kcal/mol greater than its contribution to ground state stabilization. Thus, like many residue-substrate interactions, this residue to residue interaction enhances catalysis by becoming stronger as the reaction approaches the transition state.
Alanine↗
Replacing a surface loop endows ribonuclease A with angiogenic activity.
Angiogenin (ANG) promotes the formation of blood vessels in animals. This hormone is a small, monomeric protein that is homologous to bovine pancreatic ribonuclease A (RNase). ANG is a poor ribonuclease but its ribonucleolytic activity is essential for its angiogenic activity. RNase is not angiogenic. A hybrid protein was produced in which 13 residues of a divergent surface loop of ANG were substituted for the analogous 15 residues of RNase. The value of kcat/Km for the cleavage of uridylyl(3'-->5')adenosine by this hybrid protein was 20-fold less than that of RNase but 10(5)-fold greater than that of ANG. The thermal stability of the hybrid protein was also less than that of RNase. Nevertheless, the RNase/ANG hybrid protein promotes angiogenesis in mice at least as extensively as does authentic ANG. Thus we present a protein endowed with a noncognate biological activity simply by replacing a single element of secondary structure. In addition, a 13-residue peptide corresponding to the surface loop of ANG inhibits endogenous angiogenesis in mice. These results support a model in which both a surface loop and a catalytic site are necessary for the promotion of blood vessel formation by ANG or RNase. The dissection of structure/function elements in ANG reveals a unique opportunity to develop new molecules that modulate neovascularization.
Amino Acid Sequence↗
Catalytic activity of bovine seminal ribonuclease is essential for its immunosuppressive and other biological activities.
Bovine seminal ribonuclease (BS-RNase) is a homologue of RNase A with special biological properties, including potent immunosuppressive activity. A mutant BS-RNase was created in which His-119, the active-site residue that acts as a general acid during catalysis, was changed to an aspartic acid. H119D BS-RNase formed a dimer with quaternary structure similar to that of the wild-type enzyme but with values of kcat. and kcat./Km for the cleavage of UpA [uridylyl(3'-->5')adenosine] that were 4 x 10(3)-fold lower. The mutant protein also demonstrated dramatically decreased immunosuppressive, anti-tumour, aspermatogenic, and embryotoxic activities. The catalytic activity of BS-RNase is therefore necessary for its special biological properties.
Animals↗
Structural basis for the biological activities of bovine seminal ribonuclease.
Bovine seminal ribonuclease (BS-RNase) is a homolog of RNase A with special biological properties that include specific antitumor, aspermatogenic, and immuno-suppressive activities. Unlike RNase A, BS-RNase is a dimer cross-linked by disulfide bonds between Cys31 of one subunit and Cys32 of the other. At equilibrium, this dimer is a mixture of two distinct quaternary forms, M = M and M x M. The conversion of M = M to M x M entails the exchange of NH2-terminal alpha-helices between subunits. Here, the cytotoxic activities of purified M x M were shown to be greater than those of purified M = M, despite extensive equilibration of M = M and M x M during the time course of the assays. Replacing Cys31 or Cys32 with a serine residue did not compromise the enzymatic activity of dimeric BS-RNase, but reduced both the fraction of M x M at equilibrium and the cytotoxicity. We conclude that the M x M form is responsible for the special biological properties of BS-RNase. Since cytosolic ribonuclease inhibitor binds tightly to monomeric but not dimeric BS-RNase and only the M x M form can remain dimeric in the reducing environment of the cytosol, we propose that BS-RNase has evolved its M x M form to retain its lethal enzymatic activity in vivo.
Animals↗
Dibromobimane as a fluorescent crosslinking reagent.
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Animals↗
The extent to which ribonucleases cleave ribonucleic acid.
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Glycine↗
Production of rat protein disulfide isomerase in Saccharomyces cerevisiae.
Protein disulfide isomerase (PDI) is an abundant protein of the endoplasmic reticulum that catalyzes the oxidation of protein sulfhydryl groups and the isomerization and reduction of protein disulfide bonds. Saccharomyces cerevisiae cells lacking PDI are inviable. PDI is a component of many different protein processing complexes, and the actual activity of PDI that is required for cell viability is unclear. A cDNA that codes for rat PDI fused to the alpha-factor pre-pro segment was expressed in a protease-deficient strain of S. cerevisiae under the control of an ADH2-GAPDH hybrid promoter. The cells processed the resulting protein and secreted it into the medium as a monomer, despite having a KDEL or HDEL sequence at its C-terminus. The typical yield of isolated protein was 2 mg per liter of culture. The catalytic activity of the PDI from S. cerevisiae was indistinguishable from that of PDI isolated from bovine liver. This expression system is unique in allowing the same plasmid to be used both to complement pdi1 delta S. cerevisiae and to produce PDI for detailed in vitro analyses. Correlations of the in vivo behavior and in vitro properties of PDI are likely to reveal structure-function relationships of biological importance.
Alcohol Dehydrogenase↗
Engineering ribonuclease A: production, purification and characterization of wild-type enzyme and mutants at Gln11.
Bovine pancreatic ribonuclease A (RNase A) has been the object of much landmark work in biological chemistry. Yet the application of the techniques of protein engineering to RNase A has been limited by problems inherent in the isolation and heterologous expression of its gene. A cDNA library was prepared from cow pancreas, and from this library the cDNA that codes for RNase A was isolated. This cDNA was inserted into expression plasmids that then directed the production of RNase A in Saccharomyces cerevisiae (fused to a modified alpha-factor leader sequence) or Escherichia coli (fused to the pelB signal sequence). RNase A secreted into the medium by S.cerevisiae was an active but highly glycosylated enzyme that was recoverable at 1 mg/l of culture. RNase A produced by E.coli was in an insoluble fraction of the cell lysate. Oxidation of the reduced and denatured protein produced active enzyme which was isolated at 50 mg/l of culture. The bacterial expression system is ideal for the large-scale production of mutants of RNase A. This system was used to substitute alanine, asparagine or histidine for Gln11, a conserved residue that donates a hydrogen bond to the reactive phosphoryl group of bound substrate. Analysis of the binding and turnover of natural and synthetic substrates by the wild-type and mutant enzymes shows that the primary role of Gln11 is to prevent the non-productive binding of substrate.
Amino Acid Sequence↗
A misfolded but active dimer of bovine seminal ribonuclease.
Bovine seminal ribonuclease (BS-RNase) is an unusual homolog of RNase A. Isolated from bulls as a dimer, BS-RNase has special biological properties including antispermatogenic, antitumor and immunosuppressive activities. The structural bases for these properties are unknown. Four forms of BS-RNase were isolated after folding and air oxidation of the denatured and reduced protein produced in Escherichia coli: two dimers (M = M and M x I, where x signifies an active site composed of residues from both subunits) and two monomers (M and I). Considerable ribonuclease activity was generated by air oxidation of an equimolar mixture of two inactive mutant proteins ([H12D]BS-RNase and [H119D]BS-RNase) prepared by site-directed mutagenesis. This activity came from a dimer (M x I) with a composite active site. 1H-NMR spectroscopy revealed that this dimer contained one correctly folded subunit (M), and one incorrectly folded subunit (I). Form I, which is a poor catalyst, was activated by ribonuclease S-protein, suggesting that the C-terminal portion of I was not folded properly. Electrospray-ionization mass spectrometry and sulfhydryl group titration indicated that I contains a single oxidized sulfhydryl group, which cannot participate in a disulfide bond. These results show that quaternary structure in BS-RNase is attained by the initial formation of two monomers, M and I, which then combine with another M to form M = M and M x I, respectively. Adventitious oxidation can thus lead to the formation of a misfolded but active enzyme (M x I).
Animals↗