Biomedical subjects

G H Lorimer

Publications and source records attributed to G H Lorimer.

At least 37 records · Page 2Linked to original sources

PubMed1993

Plurality of protein conformations of ribulose-1,5-bisphosphate carboxylase/oxygenase monomers probed by high pressure electrophoresis.

We used hydrostatic pressure in the range of 1 to 2 kbar, coupled with polyacrylamide gel electrophoresis, to investigate the properties of monomers of dimeric ribulose bisphosphate carboxylase/oxygenase. At temperatures below -5 degrees C or pressures above 1.5 kbar, only a diffuse band with low electrophoretic mobility was observed, which is assigned to a denatured monomer. In gels run at 1.0 kbar and temperatures above 0 degree C, both the wild type and a mutant in which a positively charged Lys at the dimer interface is replaced by a negatively charged glutamic acid displayed several discrete bands with retardation coefficients larger than that of the dimer. Cross-linking due to oxidation of cysteines was not the reason for the multiplicity of bands, which in addition were independent on the length of the electrophoretic run in the range of 1-3 h. Binding of 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid to the pressure dissociated monomers stabilized the unfolded conformations. We propose that the dissociated monomers adopt various expanded conformations, which, under the experimental conditions, are stabilized to the extent necessary to be considered as distinct chemical species. Gel filtration high performance liquid chromatography analysis of bands eluted from nonstained gels run at 1 kbar (15 degrees C) and 1.5 kbar (-5 degrees C), respectively, was performed. In both cases the dimeric structure was fully recovered, along with the spectroscopic properties and catalytic activity characteristic of the native dimer, indicating that the unresolved unfolded conformers that appear at -5 degrees C, as well as the set of discrete conformers obtained at 15 degrees C, are able to reconstitute a single active conformation on reassociation.

Electrophoresis, Polyacrylamide Gel↗

PubMed1993

Identification and functional analysis of chaperonin 10, the groES homolog from yeast mitochondria.

Chaperonin 60 (cpn60) and chaperonin 10 (cpn10) constitute the chaperonin system in prokaryotes, mitochondria, and chloroplasts. In Escherichia coli, these two chaperonins are also termed groEL and groES. We have used a functional assay to identify the groES homolog cpn10 in yeast mitochondria. When dimeric ribulose-1,5-bisphosphate carboxylase (Rubisco) is denatured and allowed to bind to yeast cpn60, subsequent refolding of Rubisco is strictly dependent upon yeast cpn10. The heterologous combination of cpn60 from E. coli plus yeast cpn10 is also functional. In contrast, yeast cpn60 plus E. coli cpn10 do not support refolding of Rubisco. In the presence of MgATP, yeast cpn60 and yeast cpn10 form a stable complex that can be isolated by gel filtration and that facilitates refolding of denatured Rubisco. Although the potassium-dependent ATPase activity of E. coli cpn60 can be inhibited by cpn10 from either E. coli or yeast, neither of these cpn10s inhibits the ATPase activity of yeast cpn60. Amino acid sequencing of yeast cpn10 reveals substantial similarity to the corresponding cpn10 proteins from rat mitochondria and prokaryotes.

Adenosine Triphosphatases↗

PubMed1993

A role for the epsilon-amino group of lysine-334 of ribulose-1,5-bisphosphate carboxylase in the addition of carbon dioxide to the 2,3-enediol(ate) of ribulose 1,5-bisphosphate.

Earlier structural and functional studies of ribulose-1,5-bisphosphate carboxylase/oxygenase imply that K334 facilitates the addition of gaseous substrate to the 2,3-enediol(ate) derived from ribulose 1,5-bisphosphate. Crystallographic analysis of the activated spinach enzyme [Knight et al. (1990) J. Mol. Biol. 215, 113-160] shows that the lysyl side chain is appropriately positioned to stabilize the transition state for the addition of CO2 to the enediol(ate). Furthermore, despite total impairment of carboxylase and oxygenase activities, site-directed mutants of the Rhodospirillum rubrum enzyme with replacements for lysine K334 (formerly designated K329) retain the capacity to enolize ribulose bisphosphate, demonstrating that the primary catalytic lesion lies beyond this initial step [Soper et al. (1988) Protein Eng. 2, 39-44; Hartman & Lee (1989) J. Biol. Chem. 264, 11784-11789]. We now show that the K334C mutant is also competent in the latter stages of catalysis, whereby 2'-carboxy-3-keto-D-arabinitol 1,5-bisphosphate, the six-carbon intermediate of the carboxylation pathway, is correctly processed to 3-phosphoglycerate. Thus, the impairment of the mutant in overall catalysis can be attributed to preferential disruption of the reaction of CO2 or O2 with the enzyme-bound enediol(ate). Chemical rescue of the K334C mutant by aminoethylation and aminopropylation shows that this disruption reflects, at least in part, a failure to adequately stabilize the relevant transition state. With several simplifying assumptions, the CO2/O2 specificity factor tau can be reduced to the ratio of the fundamental second-order rate constants for the interaction of the gaseous substrates with the enzyme-bound 2,3-enediol(ate) of ribulose bisphosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1993

Hydrolysis of adenosine 5'-triphosphate by Escherichia coli GroEL: effects of GroES and potassium ion.

The potassium-ion activation constant (Kact) for the ATPase activity of Escherichia coli chaperonin groEL is inversely dependent upon the ATP concentration over at least 3 orders of magnitude. The ATPase activity shows positively cooperative kinetics with respect to ATP and K+. Both the K0.5 for ATP and cooperativity (as measured by the Hill coefficient) decrease as the K+ concentration increases. Equilibrium binding studies under conditions where hydrolysis does not occur indicate that MgATP binds weakly to groEL in the absence of K+. In the absence of groES, the K(+)-dependent hydrolysis of ATP by groEL continues to completion. In the presence of groES, the time course for the hydrolysis of ATP by groEL becomes more complex. Three distinct kinetic phases can be discerned. Initially, both heptameric toroids turn over once at the same rate that they do in the absence of groES. This leads to the formation of an asymmetric binary complex, groEL14-MgADP7-groES7, in which 7 mol of ADP is trapped in a form that does not readily exchange with free ADP. In the second phase, the remaining seven sites (containing readily exchangeable ADP) turn over, or have the potential to turn over, at the same rate as they do in the absence of groES, so that the overall rate of hydrolysis is maximally 50%. These remaining sites of the asymmetric binary complex do not hydrolyze all of the available ATP. Instead, the second phase of hydrolysis gives way to a third, completely inhibited state, the onset of which is dependent upon the relative affinities of the remaining sites for MgATP and MgADP.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗

PubMed1993

Reversible dissociation and conformational stability of dimeric ribulose bisphosphate carboxylase.

Dimer-monomer dissociation of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum was investigated using hydrostatic pressure in the range 1-2 kbar to promote dissociation. Intrinsic fluorescence emission and polarization, along with the polarization of the fluorescence of single-labeled AEDANS conjugates, were used to follow the dissociation. Full reversibility after dissociation was observed to depend on the presence of small ligands: glycerol, Mg2+, and NaHCO3, the last two being required to activate the enzyme. The free energy of association at 15 degrees C, -12.9 kcal mol-1, was made up of a positive change in enthalpy on association of 6.0 kcal mol-1 and an entropic contribution (T delta S) of 18.9 kcal mol-1; thus the monomer association is entropy driven. No dissociation of the quaternary complex formed by the dimer, 2-carboxy-D-arabinitol 1,5-diphosphate (CADP), Mg2+, and NaHCO3 was observed at pressures up to 2.0 kbar; the magnitude of stabilization by the inhibitor binding was estimated as 2.3 kcal mol-1. Pressurization in the presence of bis-ANS results in a time-dependent increase in fluorophore emission, indicating changes in monomer conformation with exposure of hydrophobic surfaces upon dissociation. Reactivity against the fluorescent probe 1,5-I-AEDANS was also used as a conformational probe: HPLC of a trypsin digest of rubisco labeled at atmospheric pressure revealed a single fluorescent peptide, whereas more extensive labeling was observed when the reaction was carried out at 2.0 kbar, indicative of exposure of internal cysteines.(ABSTRACT TRUNCATED AT 250 WORDS)

Bicarbonates↗

PubMed1993

Chaperonins and protein folding: unity and disunity of mechanisms.

Chaperonin-facilitated folding of proteins involves two partial reactions. The first partial reaction, the formation of stable binary complexes between chaperonin-60 and non-native states of the target protein, is common to the chaperonin-facilitated folding of all target proteins investigated to date. The structural basis for this interaction is not presently understood. The second partial reaction, the dissociation of the target protein in a form committed to the native state, appears to proceed by a variety of mechanisms, dependent upon the nature of the target protein in question. Those target proteins (e.g. rubisco, rhodanese, citrate synthase) which require the presence of chaperonin-10, also appear to require the hydrolysis of ATP to bring about the dissociation of the target protein from chaperonin-60. With one exception (pre-beta-lactamase) those target proteins which do not require the presence of chaperonin-10 to be released from chaperonin-60, also do not require the hydrolysis of ATP, since non-hydrolysable analogues of ATP support the release of the target protein in a state committed to the native state. The question of whether or not chaperonin-facilitated folding constitutes a catalysed event is addressed.

Animals↗

PubMed1992

Chaperonin cpn60 from Escherichia coli protects the mitochondrial enzyme rhodanese against heat inactivation and supports folding at elevated temperatures.

The chaperonin protein cpn60 from Escherichia coli protects the monomeric, mitochondrial enzyme rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) against heat inactivation. The thermal inactivation of rhodanese was studied for four different states of the enzyme: native, refolded, bound to cpn60 in the form of a binary complex formed from unfolded rhodanese, and a thermally perturbed state. Thermal stabilization is observed in a range of temperatures from 25 to 48 degrees C. Rhodanese that had been inactivated by incubation at 48 degrees C, in the presence of cpn60 can be reactivated at 25 degrees C, upon addition of cpn10, K+, and MgATP. A recovery of about 80% was achieved after 1 h of the addition of those components. Thus, the enzyme is protected against heat inactivation and kept in a reactivable form if inactivation is attempted using the binary complex formed between rhodanese folding intermediate(s) and cpn60. The chaperonin-assisted refolding of urea-denatured rhodanese is dependent on the temperature of the refolding reaction. However, optimal chaperonin assisted refolding of rhodanese observed at 25 degrees C, which is achieved upon addition of cpn10 and ATP to the cpn60-rhodanese complex, is independent of the temperature of preincubation of the complex, that was formed previously at low temperature. The results are in agreement with a model in which the chaperonin cpn60 interacts with partly folded intermediates by forming a binary complex which is stable to elevated temperatures. In addition, it appears that native rhodanese can be thermally perturbed to produce a state different from that achieved by denaturation that can interact with cpn60.

Bacterial Proteins↗

PubMed1992

Conformational states of ribulosebisphosphate carboxylase and their interaction with chaperonin 60.

Conformational states of ribulosebisphosphate carboxylase (Rubisco) from Rhodospirillum rubrum were examined by far-UV circular dichroism (CD), tryptophan fluorescence, and 1-anilino-naphthalenesulfonate (ANS) binding. At pH 2 and low ionic strength (I = 0.01), Rubisco adopts an unfolded, monomeric conformation (UA1 state) as judged by far-UV CD and tryptophan fluorescence. As with other acid-unfolded proteins [Goto, Y., Calciano, L. J., & Fink, A. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 573-577], an intermediate conformation (A1 state) is observed at pH 2 and high ionic strength. The A1 state has an alpha-helical content equivalent to 64% of that present in the native dimer (N2 state). However, fluorescence measurements indicate that the tertiary structure of the A1 state is largely disordered. A site-directed mutant, K168E, which exists as a stable monomer [Mural, R. J., Soper, T. S., Larimer, F. W., & Hartman, F. C. (1990) J. Biol. Chem. 265, 6501-6505] was used to characterize the "native" monomer (N1 state). The far-UV CD spectra of the N1 and N2 states are almost identical, indicating a similar secondary structure content. However, the tertiary structure of the N1 state is less ordered than that of the N2 state. Nevertheless, when appropriately complemented in vitro, K168E forms an active heterodimer. Upon neutralization of acid-denatured Rubisco or dilution of guanidine hydrochloride-denatured Rubisco, unstable folding intermediates (I1 state) are rapidly formed. At concentrations at or below the "critical aggregation concentration" (CAC), the I1 state reverts spontaneously but slowly to the native states with high yield (greater than 65%). The CAC is temperature-dependent. At concentrations above the CAC, the I1 and the A1 states undergo irreversible aggregation. The commitment to aggregation is rapid [ef. Goldberg, M. E., Rudolph, R., & Jaenicke, R. (1991) Biochemistry 30, 2790-2797] and proceeds until the concentration of folding intermediate(s) has fallen to the CAC. In the presence of a molar excess of chaperonin 60 oligomers, the I1 state forms a stable binary complex. No stable binary complex between chaperonin 60 and the N1 state could be detected. Formation of the chaperonin 60-I1 binary complex arrests the spontaneous folding process. The I1 state becomes resistant to interaction with chaperonin 60 with kinetics indistinguishable from those associated with the appearance of the native states. In vitro complementation analysis indicated that the product of the chaperonin-facilitated process is monomeric.(ABSTRACT TRUNCATED AT 400 WORDS)

Anilino Naphthalenesulfonates↗

PubMed1992

Mammalian mitochondrial chaperonin 60 functions as a single toroidal ring.

Chaperonins are thought to participate in the process of protein folding in bacteria and in eukaryotic mitochondria and chloroplasts. While some chaperonins are relatively well characterized, the structures of the mammalian chaperonins are unknown. We have expressed a mammalian mitochondrial chaperonin 60 in Escherichia coli and purified the recombinant protein to homogeneity. Structural and biochemical analyses of this protein establish a single toroidal structure of seven subunits, in contrast to the homologous bacterial, fungal, and plant chaperonin 60s, which have double toroidal structures comprising two layers of seven identical subjects each. The recombinant mammalian chaperonin 60, together with the mammalian chaperonin 10 (but not with bacterial chaperonin 10), facilitates the formation of catalytically active ribulose-bisphosphate carboxylase from an unfolded state in the presence of K+ and MgATP. Analysis of the partial reactions involved in this in vitro reconstitution reveals that the single toroid of chaperonin 60 can form stable complexes with both unfolded or partially folded [35S]ribulose-bisphosphate carboxylase and mitochondrial (but not bacterial) chaperonin 10 in the presence of MgATP. We conclude that the minimal functional unit of chaperonin 60 is a single hepatmeric toroid.

Adenosine Triphosphate↗

PubMed1992

Purified chaperonin 60 (groEL) interacts with the nonnative states of a multitude of Escherichia coli proteins.

In vitro experiments employing the soluble proteins from Escherichia coli reveal that about half of them, in their unfolded or partially folded states, but not in their native states, can form stable binary complexes with chaperonin 60 (groEL). These complexes can be isolated by gel filtration chromatography and are efficiently discharged upon the addition of Mg.ATP. Binary complex formation is substantially reduced if chaperonin 60 is presaturated with Rubisco-I, the folding intermediate of Rubisco, but not with native Rubisco. Binary complex formation is also reduced if the transient species that interact with chaperonin 60 are permitted to progress to more stable states. This implies that the structural elements or motifs that are recognized by chaperonin 60 and that are responsible for binary complex formation are only present or accessible in the unfolded states of proteins or in certain intermediates along their respective folding pathways. Given the high-affinity binding that we have observed in the present study and the normal cellular abundance of chaperonin 60, we suspect that the folding of most proteins in E. coli does not occur in free solution spontaneously, but instead takes place while they are associated with molecular chaperones.

Bacterial Proteins↗

PubMed1991

Complex interactions between the chaperonin 60 molecular chaperone and dihydrofolate reductase.

The spontaneous refolding of chemically denatured dihydrofolate reductase (DHFR) is completely arrested by chaperonin 60 (GroEL). This inhibition presumably results from the formation of a stable complex between chaperonin 60 and one or more intermediates in the folding pathway. While sequestered on chaperonin 60, DHFR is considerably more sensitive to proteolysis, suggesting a nonnative structure. Bound DHFR can be released from chaperonin 60 with ATP, and although chaperonin 10 (GroES) is not obligatory, it does potentiate the maximum effect of ATP. Hydrolysis of ATP is also not required for DHFR release since certain nonhydrolyzable analogues are capable of partial discharge. "Native" DHFR can also form a stable complex with chaperonin 60. However, in this case, complex formation is not instantaneous and can be prevented by the presence of DHFR substrates. This suggests that native DHFR exists in equilibrium with at least one conformer which is recognizable by chaperonin 60. Binding studies with 35S-labeled DHFR support these conclusions and further demonstrate that DHFR competes for a common saturable site with another protein (ribulose-1,5-bisphosphate carboxylase) known to interact with chaperonin 60.

Adenosine Triphosphate↗

PubMed1991

Intermediates in the chaperonin-assisted refolding of rhodanese are trapped at low temperature and show a small stoichiometry.

In vitro refolding of the urea-unfolded, monomeric, mitochondrial enzyme rhodanese (thiosulfate sulfur-transferase; EC 2.8.1.1) is facilitated by the chaperonin proteins cpn60 and cpn10 from Escherichia coli at 37 degrees C, but the refolding is strongly inhibited at 10 degrees C. In contrast, the unassisted refolding of rhodanese is efficient at 10 degrees C, but the refolding efficiency decreases as the temperature is raised. These observations provided two measures of the cpn60-rhodanese complex. Thus, we monitored either 1) the cpn60-dependent inhibition of spontaneous folding at 10 degrees C or 2) the recovery of active rhodanese in the complete chaperonin system at 25 degrees C, after first forming a cpn60-rhodanese complex at 10 degrees C. These procedures minimized the aggregation of interactive folding intermediates that tend to overestimate the apparent number of cpn60 14-mers in determining the stoichiometry of protein-cpn60 14-mer interactions. Both procedures used here gave results that were consistent with there being 1 rhodanese binding site/cpn60 tetradecamer. This stoichiometry is significantly less than might be expected from the fact that cpn60 is composed of 14 identical subunits, and it may indicate that rhodanese interacts with a restricted region that is formed when the cpn60 tetradecamer is assembled. The ability to stabilize chaperonin-protein complexes that can subsequently be reactivated will aid studies of the mode of action of the ubiquitous chaperonin proteins.

Adenosine Triphosphate↗

PubMed1991

Unassisted refolding of urea unfolded rhodanese.

In vitro refolding after urea unfolding of the enzyme rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) normally requires the assistance of detergents or chaperonin proteins. No efficient, unassisted, reversible unfolding/folding transition has been demonstrated to date. The detergents or the chaperonin proteins have been proposed to stabilize folding intermediates that kinetically limit folding by aggregating. Based on this hypothesis, we have investigated a number of experimental conditions and have developed a protocol for refolding, without assistants, that gives evidence of a reversible unfolding transition and leads to greater than 80% recovery of native enzyme. In addition to low protein concentration (10 micrograms/ml), low temperatures are required to maximize refolding. Otherwise optimal conditions give less than 10% refolding at 37 degrees C, whereas at 10 degrees C the recovery approaches 80%. The unfolding/refolding phases of the transition curves are most similar in the region of the transition, and refolding yields are significantly reduced when unfolded rhodanese is diluted to low urea concentrations, rather than to concentrations near the transition region. This is consistent with the formation of "sticky" intermediates that can remain soluble close to the transition region. Apparently, nonnative structures, e.g. aggregates, can form rapidly at low denaturant concentrations, and their subsequent conversion to the native structure is slow.

In Vitro Techniques↗

PubMed1991

Chaperonins facilitate the in vitro folding of monomeric mitochondrial rhodanese.

In vitro refolding of the monomeric mitochondrial enzyme, rhodanese (thiosulfate sulfurtransferase; EC 2.8.1.1) is facilitated by molecular chaperonins. The four components: two proteins from Escherichia coli, chaperonin 60 (groEL) and chaperonin 10 (groES), MgATP, and K+, are necessary for the in vitro folding of rhodanese. These were previously shown to be necessary for the in vitro folding of ribulose-1,5-bisphosphate carboxylase at temperatures in excess of 25 degrees C (Viitanen, P. V., Lubben, T. H., Reed, J., Goloubinoff, P., O'Keefe, D. P., and Lorimer, G. H. (1990) Biochemistry 29, 5665-5671). The labile folding intermediate, rhodanese-I, which rapidly aggregates at 37 degrees C in the absence of the chaperonins, can be stabilized by forming a binary complex with chaperonin 60. The discharge of the binary chaperonin 60-rhodanese-I complex, results in the formation of active rhodanese, and requires the presence of chaperonin 10. Optimal refolding is associated with a K(+)-dependent hydrolysis of ATP. At lower protein concentrations and 25 degrees C, where aggregation is reduced, a fraction of the rhodanese refolds to an active form in the absence of the chaperonins. This spontaneous refolding can be arrested by chaperonin 60. There is some refolding (approximately equal to 20%) when ATP is replaced by nonhydrolyzable analogs, but there is no refolding in the presence of ADP or AMP. ATP analogs may interfere with the interaction of rhodanese-I with the chaperonins. Nondenaturing detergents facilitate rhodanese refolding by interacting with exposed hydrophobic surfaces of folding intermediates and thereby prevent aggregation (Tandon, S., and Horowitz, P. (1986) J. Biol. Chem. 261, 15615-15618). The chaperonin proteins appear to play a similar role in as much as they can replace the detergents. Consistent with this view, chaperonin 60, but not chaperonin 10, binds 2-3 molecules of the hydrophobic fluorescent reporter, 1,1'-bi(4-anilino)naphthalene-S,5'-disulfonic acid, indicating the presence of hydrophobic surfaces on chaperonin 60. The number of bound probe molecules is reduced to 1-2 molecules when chaperonin 10 and MgATP are added. The results support a model in which chaperonins facilitate folding, at least in part, by interacting with partly folded intermediates, thus preventing the interactions of hydrophobic surfaces that lead to aggregation.

Adenosine Triphosphate↗

PubMed1990

Chaperonin-facilitated refolding of ribulosebisphosphate carboxylase and ATP hydrolysis by chaperonin 60 (groEL) are K+ dependent.

Both the chaperonin- and MgATP-dependent reconstitution of unfolded ribulosebisphosphate carboxylase (Rubisco) and the uncoupled ATPase activity of chaperonin 60 (groEL) require ionic potassium. The spontaneous, chaperonin-independent reconstitution of Rubisco, observed at 15 but not at 25 degrees C, requires no K+ and is actually inhibited by chaperonin 60, with which the unfolded or partly folded Rubisco forms a stable binary complex. The chaperonin-dependent reconstitution of Rubisco involves the formation of a complex between chaperonin 60 and chaperonin 10 (groES). Formation of this complex almost completely inhibits the uncoupled ATPase activity of chaperonin 60. Furthermore, although the formation of the chaperonin 60-chaperonin 10 complex requires the presence of MgATP, hydrolysis of ATP may not be required, since complex formation occurs in the absence of K+. The interaction of chaperonin 60 with unfolded or partly folded Rubisco does not require MgATP, K+, or chaperonin 10. However, discharge of the complex of chaperonin 60-Rubisco, which leads to the formation of active Rubisco dimers, requires chaperonin 10 and a coupled, K(+)-dependent hydrolysis of ATP. We propose that a role of chaperonin 10 is to couple the K(+)-dependent hydrolysis of ATP to the release of the folded monomers of the target protein from chaperonin 60.

Adenosine Triphosphatases↗

PubMed1990

Chaperonin assisted polypeptide folding and assembly: implications for the production of functional proteins in bacteria.

Production of biologically active foreign proteins with correct three-dimensional structures is often difficult in bacteria. Recent advances demonstrate that, for some proteins at least, their correct folding and assembly is facilitated by a class of proteins known as molecular chaperones. An understanding of the function of molecular chaperones may assist in the synthesis in bacteria of functional foreign proteins produced by recombinant techniques.

Bacteria↗

PubMed1990

Identification of a groES-like chaperonin in mitochondria that facilitates protein folding.

Mitochondria contain a polypeptide that is functionally equivalent to Escherichia coli chaperonin 10 (cpn10; also known as groES). This mitochondrial cpn10 has been identified in beef and rat liver and is able to replace bacterial cpn10 in the chaperonin-dependent reconstitution of chemically denatured ribulose-1,5-bisphosphate carboxylase. Thus, like the bacterial homologue, mitochondrial cpn10 facilitates a K(+)- and Mg.ATP-dependent discharge of unfolded (or partially folded) ribulose bisphosphate carboxylase from bacterial chaperonin 60 (cpn60; also known as groEL). Instrumental to its identification, mitochondrial cpn10 and bacterial cpn60 form a stable complex in the presence of Mg.ATP. Bacterial and mitochondrial cpn10 compete for a common saturable site on bacterial cpn60. As a result of complex formation, with either mitochondrial or bacterial cpn10, the "uncoupled ATPase" activity of bacterial cpn60 is virtually abolished. The most likely candidate for mitochondrial cpn10 is an approximately 45-kDa oligomer composed of approximately 9-kDa subunits. We propose that, like the protein-folding machinery of prokaryotes, mitochondrial cpn60 requires a cochaperonin for full biological function.

Adenosine Triphosphate↗

PubMed1989

The orientation of substrate and reaction intermediates in the active site of ribulose-1,5-bisphosphate carboxylase.

There are four possible orientations of the substrate ribulose 1,5-bisphosphate in the active site of ribulose-1,5-bisphosphate carboxylase. Distinction between these four possible orientations has been made on the basis of 31P NMR and borohydride-trapping experiments. The orientation of the reaction-intermediate analog, 2'-carboxy-D-arabinitol 1,5-bisphosphate with respect to the divalent metal ion was determined by 31P NMR studies of the quaternary complex, enzyme.CO2.Ni2+.2'-carboxyarabinitol 1,5-bisphosphate. Assignment of the phosphorus resonances of this complex was made by labeling the phosphoryl group at either C-1 or C-5 with 17O. The phosphorus atom closer to the paramagnetic metal ion, Ni2+, to which the broader of the phosphorus resonances is attributed, has been identified as that attached to C-1. When bound to the active site of carbaminated enzyme, D-ribulose 1,5-bisphosphate was reduced by sodium borohydride with absolute stereospecificity to D-arabinitol 1,5-bisphosphate. The reduction of the enzyme-bound substrate thus occurred on the Si face of the C-2 carbonyl group. These two results together establish that ribulose 1,5-bisphosphate is oriented within the active site so that 1) the phosphoryl group at C-1 is closer to the divalent metal ion than that at C-5 and 2) the Si face of the carbonyl group points to the "outside world."

Binding Sites↗