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PubMed1991

Substrate regulation of single potassium and chloride ion channels in Arabidopsis plasma membrane.

Patch clamp measurements of excised inside-out patches of Arabidopsis thaliana plasma membrane reveal at least two ion channels which conduct either potassium or chloride. The conductance of the potassium channel ranged from 5 to 70 picosiemens depending on KCl concentration. The conductance increased linearly with increasing cytoplasmic-side [KCl]; the extent of this dependence declined as extracytoplasmic-side [KCl] was increased. This indicates that substrate regulation of the potassium channel is a consequence of the molecular architecture of the channel: in particular, multi-ion binding sites within the channel pore. The chloride channel conductance (ranging from 5-40 picosiemens) was independent of cytoplasmic-side [KCl] until a threshold concentration of about 300 millimolar was reached. Such behavior is expected only if the channel is allosterically regulated by cytoplasmic-side K(+) and/or Cl(-). The median open times of either channel (about 200 milliseconds for the potassium channel and 20 milliseconds for the chloride channel) were unaffected by substrate concentrations.

Journal Article↗
PubMed1975

Regulation of ribulose 1,5-diphosphate carboxylase by substrates and other metabolites: further evidence for several types of binding sites.

Ribulose 1,5-diphosphate carboxylase (RuDPCase, EC 4.1.1.39) isolated from spinach leaves is metabolically regulated at 10 mm Mg(2+) and low CO(2) concentrations by its substrates (RuDP and CO(2)) and by effectors which include 6-phosphogluconate (6-PGluA), NADPH, and fructose 1,6-diphosphate (FDP), but not fructose 6-phosphate. Physiological concentrations of RuDP severely inhibit the enzyme activity when the enzyme has not been preincubated with HCO(3) (-) and Mg(2-), and this inactivity persists for 20 minutes or longer after 1 mm HCO(3) (-) and 10 mm Mg(2+) are added. Maximum activity requires that the preincubation mixture also include either 0.01 mm 6-PGluA or 0.5 mm NADPH.When the enzyme, following preincubation with HCO(3) (-) and Mg(2+), is presented with RuDP plus either 6-PGluA or FDP, competitive inhibition is observed with respect to RuDP. The Ki value for 6-PGluA is 0.02 mm and the Ki value for FDP is 190 mum. NADPH or 3-phosphoglycerate (PGA) at physiological concentrations does not have any effect when presented simultaneously with RuDP. Other studies on the order of addition of substrates and effectors, concentration effects, and kinetics provide additional information that serves as a basis for a proposed model of allosteric regulation combined with competitive inhibition.In this model, there are catalytic sites at which the substrates and 6-PGluA and FDP can bind, and at least four allosteric regulatory sites, which we designate I, A(1), A(2), and A(3). RuDP binds very tightly to site I (in the absence of Mg(2+) or HCO(3) (-)), causing a conformational change in the protein to an inactive form which persists for as long as 20 minutes in the subsequent presence of Mg(2+) and 1 mm HCO(3) (-). Mg(2+) and HCO(3) (-) (or CO(2)) bind to site A(3) (in the absence of RuDP), holding the enzyme in an active form which has a much lower affinity for RuDP at site I, so that when physiological levels of RuDP are then added, only part of the enzyme activity is lost. This active form of the enzyme can bind 6-PGluA or FDP at site A(1) and NADPH at site A(2) during preincubation with Mg(2+) and HCO(3) (-). With optimal levels of bound effectors, 6-PGluA or NADPH, enzyme activity is fully maintained, even when RuDP is subsequently added. Without one of these effectors present, addition of RuDP following preincubation reduces enzyme activity to about 40% at the levels of substrates and effectors studied. FDP is a much poorer effector, and this is ascribed to a possible binding of FDP at site I, as well as at site A(1).The physiological role of this regulation is discussed, particularly with respect to protection of "C-3" plants against oxidation of RuDP to phosphoglycolate.

Journal Article↗
PubMed1989

Sequence and characteristics of the Bifidobacterium longum gene encoding L-lactate dehydrogenase and the primary structure of the enzyme: a new feature of the allosteric site.

The gene ldh, encoding L-lactate dehydrogenase (LDH; EC 1.1.1.27) of Bifidobacterium longum aM101-2, was cloned in Escherichia coli using an oligodeoxyribonucleotide hybridization probe. The amino acid (aa) sequence, deduced from the sequence of the cloned DNA, was consistent with the results of protein chemical analysis of B. longum LDH. The transcription start points (tsp) in B. longum were identified by S1 nuclease mapping. A sequence, GTAGCAA-(14 bp)-TTATAGA, which is located a few bp upstream from the tsp, was assigned as the promoter of this ldh gene. In the 3'-noncoding region, there were two structures that strongly resembled the Rho-independent transcriptional termination signal of E. coli. Therefore, the B. longum ldh gene might form a monocistronic unit. The deduced primary structure of B. longum LDH had 40% identity with LDHs from Thermus caldophilus, Bacillus stearothermophilus, Lactobacillus casei and dogfish muscle. Most bacterial LDHs are allosterically regulated by fructose 1,6-bisphosphate (FBP), while the vertebrate LDHs are not. The anion-binding site of vertebrate LDHs has been thought to correspond to the FBP-binding site of bacterial LDHs. Although the B. longum LDH was regulated by FBP, the charge properties of aa residues in the putative FBP-binding site of the LDH were closer to those of the vertebrate LDHs than to those of bacterial LDHs.

Allosteric Site↗
PubMedDate not supplied

New aspects in the pathophysiology of cutaneous melanoma: a review of the role of thioproteins and the effect of nitrosoureas.

The importance of thioproteins, essential to the ribonucleotide reduction pathway, has been demonstrated in human primary and metastatic melanoma tissues. The thioredoxin reductase/thioredoxin and the glutathione reductase/glutathione/glutaredoxin electron transfer pathways represent alternative electron donors for ribonucleotide reductase and regulate the synthesis of deoxyribonucleotides, the substrates for DNA synthesis, in the S phase of the cell cycle. In addition to their important role in DNA synthesis and cell division, these thioproteins provide effective antioxidant defence against oxygen radicals and hydrogen peroxide. In human metastatic melanoma cells and tissues the thioredoxin reductase/thioredoxin system is located both in the cell cytosol and on plasma membranes and is under allosteric regulation by calcium. As a consequence, calcium plays an important role in determining the intracellular redox status, cell division and differentiation. Recently, the intracellular redox conditions have been shown to be important in the reaction of alkylating anti-tumour drugs such as the chloroethylnitrosoureas. In addition to previously established mechanisms, these highly reactive drugs inhibit thioredoxin reductase, glutathione reductase and ribonucleotide reductase by chloroethylation of their respective thiolate active sites. Incorporation of the 14C chloroethyl group in drug sensitive and resistant human metastatic melanoma cell lines depends on the redox status, with resistant cells being more oxic than sensitive cells. Thioredoxin reductase is 500-fold more sensitive than glutathione reductase to the newly developed nitrosourea, Fotemustine (diethyl-1-[3,2 chloroethyl]-3-nitrosoureido ethyl phosphonate). It has been shown that melanomas which respond to Fotemustine therapy contain more thioredoxin reductase whereas resistant metastases yielded the opposite result.(ABSTRACT TRUNCATED AT 250 WORDS)

Antioxidants↗
PubMed2005

Coupled calcium release channels and their regulation by luminal and cytosolic ions.

Contraction in skeletal and cardiac muscle occurs when Ca(2+) is released from the sarcoplasmic reticulum (SR) through ryanodine receptor (RyR) Ca(2+) release channels. Several isoforms of the RyR exist throughout the animal kingdom, which are modulated by ATP, Ca(2+) and Mg(2+) in the cytoplasm and by Ca(2+) in the lumen of the SR. This review brings to light recent findings on their mechanisms of action in the mammalian isoforms RyR-1 and RyR-2 with an emphasis on RyR-1 from skeletal muscle. Cytoplasmic Mg(2+) is a potent RyR antagonist that binds to two classes of cytoplasmic site, identified as low-affinity, non-specific inhibition sites and high-affinity Ca(2+) activation sites (A-sites). Mg(2+) inhibition at the A-sites is very sensitive to the cytoplasmic and luminal milieu. Cytoplasmic Ca(2+), Mg(2+) and monovalent cations compete for the A-sites. In isolated RyRs, luminal Ca(2+) alters the Mg(2+) affinity of the A-site by an allosteric mechanism mediated by luminal sites. However, in close-packed RyR arrays luminal Ca(2+) can also compete with cytoplasmic ions for the A-site. Activation of RyRs by luminal Ca(2+) has been attributed to either Ca(2+) feedthrough to A-sites or to Ca(2+) regulatory sites on the luminal side of the RyR. As yet there is no consensus on just how luminal Ca(2+) alters RyR activation. Recent evidence indicates that both mechanisms operate and are likely to be important. Allosteric regulation of A-site Mg(2+) affinity could trigger Ca(2+) release, which is reinforced by Ca(2+) feedthrough.

Adenosine Triphosphate↗
PubMed2006

Direct binding of translation initiation factor eIF2gamma-G domain to its GTPase-activating and GDP-GTP exchange factors eIF5 and eIF2B epsilon.

The GTP-binding eukaryotic translation initiation factor eIF2 delivers initiator methionyl-tRNA to the 40 S ribosomal subunit. The factor eIF5 stimulates hydrolysis of GTP by eIF2 upon AUG codon recognition, whereas the factor eIF2B promotes guanine nucleotide exchange on eIF2 to recycle the factor for additional rounds of translation initiation. The GTP-binding (G) domain resides in the gamma subunit of the heterotrimeric eIF2; however, only eIF2beta, and not eIF2gamma, has been reported to directly bind to eIF5 or eIF2B. Using proteins expressed in yeast or recombinant systems we show that full-length yeast eIF2gamma, as well as its isolated G domain, binds directly to eIF5 and the epsilon subunit of eIF2B, and we map the interaction sites to the catalytically important regions of these factors. Consistently, an internal deletion of residues 50-100 of yeast eIF5 impairs the interaction with recombinant eIF2gamma-G domain and abolishes the ability of eIF5 to stimulate eIF2 GTPase activity in translation initiation complexes in vitro. Thus, rather than allosterically regulating eIF2gamma-G domain function via eIF2beta, our data support a model in which the GTPase-activating factor eIF5 and the guanine-nucleotide exchange factor eIF2B modulate eIF2 function through direct interactions with the eIF2gamma-G domain.

Allosteric Site↗
PubMed2001

Structural insights into the ligand binding domains of membrane bound guanylyl cyclases and natriuretic peptide receptors.

Membrane bound guanylyl cyclases are single chain transmembrane receptors that produce the second messenger cGMP by either intra- or extracellular stimuli. This class of type I receptors contain an intracellular catalytic guanylyl cyclase domain, an adjacent kinase-like domain and an extracellular ligand binding domain though some receptors have their ligands yet to be identified. The most studied member is the atrial natriuretic peptide (ANP) receptor, which is involved in blood pressure regulation. Extracellular ANP binding induces a conformational change thereby activating the pre-oligomerized receptor leading to the production of cGMP. The recent crystal structure of the dimerized hormone binding domain of the ANP receptor provides a first three-dimensional view of this domain and can serve as a basis to structurally analyze mutagenesis, cross-linking, and genetic studies of this class of receptors as well as a non-catalytic homolog, the clearance receptor. The fold of the ligand binding domain is that of a bilobal periplasmic binding protein (PBP) very similar to that of the Leu/Ile/Val binding protein, AmiC, multi-domain transmembrane metabotropic glutamate receptors, and several DNA binding proteins such as the lactose repressor. Unlike these structural homologs, the guanylyl cyclase receptors bind much larger molecules at a site seemingly remote from the usual small molecule binding site in periplasmic binding protein folds. Detailed comparisons with these structural homologs offer insights into mechanisms of signal transduction and allosteric regulation, and into the remarkable usage of the periplasmic binding protein fold in multi-domain receptors/proteins.

Allosteric Site↗
PubMed2002

A functional analysis of the allosteric nucleotide monophosphate binding site of carbamoyl phosphate synthetase.

The catalytic activity of carbamoyl phosphate synthetase (CPS) from Escherichia coli is allosterically regulated by UMP, IMP, and ornithine. Thirteen amino acids within the domain that harbors the overlapping binding sites for IMP and UMP were mutated to alanine and characterized. The four residues that interact directly with the phosphate moiety of IMP in the X-ray crystal structure (K954, T974, T977, and K993) were shown to have the greatest impact on the dissociation constants for the binding of IMP and UMP and the associated allosteric effects on the kinetic constants of CPS. Of the four residues that interact with the ribose moiety of IMP (S948, N1015, T1017, and S1026), S1026 was shown to be more important for the binding of IMP than UMP. Five residues (V994, I1001, D1025, V1028, and I1029) were mutated in the region of the allosteric domain that surrounds the hypoxanthine ring of IMP. With the exception of V994A, these mutations had a modest influence on the binding and subsequent allosteric effects by UMP and IMP.

Adenosine Triphosphate↗
PubMed1979

Molecular mechanisms regulating the interactions between the benzodazepines and GABA receptors in the central nervous system.

Using radioreceptor assay techniques to measure the kinetics of GABA and diazepam receptors, a relationship between GABA and benzodiazepine receptors has been firmly established in membranes of brain and neuroblastoma NB2a clonal cell lines. Occupancy of benzodiazepine receptors uncovers a new population of GABA receptors (GABA2 receptors) endowed with high affinity for GABA. Moreover, stimulation of GABA receptors increases the affinity of 1,4-benzodiazepine receptors for 1,4-benzodiazepines. This reciprocal interaction appears to be mediated by an endogenous regulatory protein (for details on this protein see [14 and 29]) which allosterically regulates GABA2 receptors while it competitively interacts with benzodiazepines for their specific binding sites. The rank order of potency of the various 1,4-benzodiazepines to block the action of this protein inhibitor on GABA receptors is related to their capacity to displace 3H-diazepam binding. These data suggest that the interaction between the 1,4-benzodiazepine receptors and the endogenous protein modulator of GABA2 receptors might play a role in the pharmacological action of the 1,4-benzodiazepines.

Animals↗
PubMed1998

Phosphoinositide 3-kinase: the key switch mechanism in insulin signalling.

Insulin plays a key role in regulating a wide range of cellular processes. However, until recently little was known about the signalling pathways that are involved in linking the insulin receptor with downstream responses. It is now apparent that the activation of class 1a phosphoinositide 3-kinase (PI 3-kinase) is necessary and in some cases sufficient to elicit many of insulin's effects on glucose and lipid metabolism. The lipid products of PI 3-kinase act as both membrane anchors and allosteric regulators, serving to localize and activate downstream enzymes and their protein substrates. One of the major ways these lipid products of PI 3-kinase act in insulin signalling is by binding to pleckstrin homology (PH) domains of phosphoinositide-dependent protein kinase (PDK) and protein kinase B (PKB) and in the process regulating the phosphorylation of PKB by PDK. Using mechanisms such as this, PI 3-kinase is able to act as a molecular switch to regulate the activity of serine/threonine-specific kinase cascades important in mediating insulin's effects on endpoint responses.

Animals↗
PubMed1992

Mechanisms of amplification, deactivation, and noise reduction in invertebrate photoreceptors.

In this review we have discussed the problem of deactivation at both the rhodopsin and G protein levels. Of particular interest is the novel observation that rhodopsin deactivation can be modulated by light. This modulation is likely to play an important role in light adaptation by reducing the gain of transduction. One interesting possibility is that this modulation involves the phosphorylation of an arrestin-like molecule, but this remains to be tested. One of the experimental advantages of Limulus photoreceptors is the large size of the single photon responses and the fact that even single G proteins produce a detectable response. This made possible the observation that nonhydrolyzable GTP analogues produce discrete transient events rather than the step-like events that would be predicted by previous models. This observation led us to a new view of how enzyme deactivation is coupled to GTP hydrolysis on G protein. According to this view, enzymes are activated by G protein, but can be deactivated by processes that are not dependent on G protein or the hydrolysis of GTP. We have conducted several types of experiments, including some on the vertebrate rod system, that strongly support this hypothesis. A second major theme of this review is transduction noise. The available biochemical evidence suggests that both G protein and G protein-activated enzymes are likely to become spontaneously active and generate undesirable noise. Our measurements indicate, however, that this noise is orders of magnitude smaller than would be predicted by simple models, suggesting that special mechanisms must exist for suppressing this noise. We have proposed a specific mechanism by which enzymes regulated allosterically by multiple subunits could act as coincidence detectors to reduce transduction noise. Finally, there is the fundamental question of which second messengers have a direct role in invertebrate phototransduction. After Fesenko et al. (1985) showed that the light-dependent conductance in vertebrate rods was modulated by cGMP and not by Ca2+, there was rapid progress in understanding the vertebrate photoreceptor transduction mechanism. Now that it has been established that invertebrate light-dependent channels are regulated by cGMP and not by Ca2+, we can expect rapid progress in understanding invertebrate phototransduction. A key question that needs to be answered is whether the InsP3-Ca2+ pathway somehow triggers changes in cGMP or whether there is an altogether different pathway by which cGMP metabolizing enzymes are affected by light.

Adaptation, Physiological↗
PubMed2005

Maximal activation of skeletal muscle thin filaments requires both rigor myosin S1 and calcium.

The regulation by calcium and rigor-bound myosin-S1 of the rate of acceleration of 2'-deoxy-3'-O-(N-methylanthraniloyl)ADP (mdADP) release from myosin-mdADP-P(i) by skeletal muscle thin filaments (reconstituted from actin-tropomyosin-troponin) was measured using double mixing stopped-flow fluorescence with the nucleotide substrate 2'-deoxy-3'-O-(N-methylanthraniloyl). The predominant mechanism of regulation is the acceleration of product dissociation by a factor of approximately 200 by thin filaments in the fully activated conformation (bound calcium and rigor S1) relative to the inhibited conformation (no bound calcium or rigor S1). In contrast, only 2-3-fold regulation is due to a change in actin affinity such as would be expected by "steric blocking" of the myosin binding site of the thin filament by tropomyosin. The binding of one ligand (either calcium or rigor-S1) produces partial activation of the rate of product dissociation, but the binding of both is required to maximally accelerate product dissociation to a rate similar to that obtained with F-actin in the absence of regulatory proteins. The data support an allosteric regulation model in which the binding of either calcium or rigor S1 alone to the thin filament shifts the equilibrium in favor of the active conformation, but full activation requires binding of both ligands.

Actin Cytoskeleton↗
PubMed1996

Magnesium activated adenosine formation in intact perfused heart: predominance of ecto 5'-nucleotidase during hypermagnesemia.

Magnesium ion is an allosteric effector of 5'-nucleotidase and thus activates adenosine production from AMP. Two distinct 5'-nucleotidase systems, the membrane-bound ecto and the soluble cytosolic isoforms, exist in mammalian myocardium. The aim of this study was to delineate the contributions of the ecto vs. cytosolic isoforms to Mg2+-stimulated cardiac purine nucleoside formation and release. Isolated guinea pig hearts were retrogradely perfused at their physiological aortic pressure with Krebs-Henseleit bicarbonate buffer fortified with 10 mM glucose. AMP and the adenylate degradatives adenosine and inosine were measured in coronary venous effluent and in epicardial transudate, which was sampled to estimate concentrations of adenylate degradatives in the interstitium. When perfusate Mg2+ was increased from 0.6 to 6 mM, coronary vascular resistance and spontaneous heart rate fell, and steady-state coronary venous release of adenosine + inosine rose severalfold. Cytosolic free magnesium, as estimated by 31P-NMR after 15 min of perfusion with 6 mM Mg2+ or from chemically measured indicator metabolites after 30 min, rose 60 and 144% respectively (P < 0.05). Excess Mg2+ stimulated purine nucleoside release nearly threefold in coronary venous effluent and four- to sevenfold in epicardial transudate. 50 microM, alpha,beta-methylene adenosine 5'-diphosphate (AOPCP), a selective inhibitor of ecto 5'-nucleotidase, elevated interstitial AMP concentration tenfold, did not attenuate basal nucleoside release, but completely inhibited Mg2+-stimulated coronary venous purine nucleoside release and blunted Mg2+-stimulated interstitial purine nucleoside formation by 69%. During perfusion with exogenous 1 microM [8-14C]AMP, excess perfusate MgCl2 increased [14C]adenosine release by 63% in coronary effluent and 133% in epicardial transudate. AOPCP decreased baseline [14C]adenosine release in coronary effluent and epicardial transudate by 85-90%, caused equilibration of arterial and epicardial AMP, and attenuated MgCl2 activation of p[14C]adenosine formation by approx. 75%, in both the vascular and interstitial compartments. Intramyocytic concentrations of allosteric regulators of the cytosolic 5'-nucleotidases were evaluated in stop-frozen myocardium. Excess magnesium did not appreciably alter intracellular pH and ATP concentration, but lowered free cytosolic ADP and AMP concentrations by 50 and 70%, respectively. A simplified model of compartmentalized adenosine metabolism is proposed in which magnesium ion-activated cardiac purine release originates predominantly from the ecto 5'-nucleotidase; magnesium ion stimulation of metabolic flux through the cytosolic isoforms was constrained by concomitant reductions in intracellular AMP substrate and allosteric activator ADP. Magnesium ion-enhanced adenosine formation by 5'-nucleotidase could contribute to the known cardioprotective effects of this clinically used cation.

5'-Nucleotidase↗
PubMed2000

Tryptophan fluorescence reveals the conformational state of a dynamic loop in recombinant porcine fructose-1,6-bisphosphatase.

Wild-type porcine fructose-1,6-bisphosphatase (FBPase) has no tryptophan residues. Hence, the mutation of Try57 to tryptophan places a unique fluorescent probe in the structural element (loop 52-72) putatively responsible for allosteric regulation of catalysis. On the basis of steady-state kinetics, circular dichroism spectroscopy, and X-ray crystallography, the mutation has little effect on the functional and structural properties of the enzyme. Fluorescence intensity from the Trp57 mutant is maximal in the presence of divalent cations, fructose 6-phosphate and orthophosphate, which together stabilize an R-state conformation in which loop 52-72 is engaged with the active site. The level of fluorescence emission decreases monotonically with increasing levels of AMP, an allosteric inhibitor, which promotes the T-state, disengaged-loop conformation. The titration of various metal-product complexes of the Trp57 mutant with fructose 2,6-bisphosphate (F26P(2)) causes similar decreases in fluorescence, suggesting that F26P(2) and AMP individually induce similar conformational states in FBPase. Fluorescence spectra, however, are sensitive to the type of divalent cation (Zn(2+), Mn(2+), or Mg(2+)) and suggest conformations in addition to the R-state, loop-engaged and T-state, loop-disengaged forms of FBPase. The work presented here demonstrates the utility of fluorescence spectroscopy in probing the conformational dynamics of FBPase.

Amino Acid Substitution↗
PubMed2003

Modifying the oligomeric state of cyclic amidase and its effect on enzymatic catalysis.

A group of cyclic amidases, including hydantoinase, allantoinase, dihydropyrimidinase, and dihydroorotase, catalyze the reversible hydrolysis of cyclic ureides, such as 5-monosubstituted hydantoins and dihydropyrimidines. These four enzymes carry hydrophobic patches to form dimers. With the exception of dihydroorotase, these enzymes are further dimerized to form tetramers by hydrophobic interactions. This leads us to speculate that the hydrophobic interaction domain may be a significant factor in the catalytic property of these oligomeric cyclic amidases, for which activities are not allosterically regulated. We generated a dimeric D-hydantoinase by mutating five residues in the hydrophobic alpha-helical interface of a tetramer and analyzed the kinetic properties of the dimeric form of D-hydantoinase. The specific activity of the dimeric D-hydantoinase corresponds to 5.3% of the activity of tetrameric D-hydantoinase. This low specific activity of the dimeric D-hydantoinase indicates that the dimeric interaction to form a tetramer has a significant effect on the catalytic activity of this non-allosteric tetramer.

Amidohydrolases↗
PubMed2006

Light regulation of the photosynthetic phosphoenolpyruvate carboxylase (PEPC) in Hydrilla verticillata.

The submersed monocot, Hydrilla verticillata (L.f.) Royle, is a facultative C(4) NADP-malic enzyme (NADP-ME) plant in which the C(4) and Calvin cycles co-exist in the same cell. Futile cycling is avoided by an intracellular separation of carboxylases between the cytosol and chloroplasts. Of the two sequenced H. verticillata phosphoenolpyruvate carboxylase (PEPC) isoforms, hvpepc3 and hvpepc4, transcript expression of the latter was substantially up-regulated during C(4) induction, especially in the light. Western blots revealed two PEPC-specific bands in C(3) and C(4) leaf extracts; the lower band dominated in the C(4) and underwent post-translational phosphorylation in the light as determined by immunological studies. This band probably represents the photosynthetic isoform, HVPEPC4, despite the lack of the C(4) signature serine (Flaveria residue 774; Hydrilla 779). In C(4) leaves, PEPC activity increased 14-fold, was enhanced by leaf exposure to light, and showed allosteric regulation. Glucose-6-phosphate acted as a positive effector, but malate was inhibitory, with I(50) values of 0.4 and 0.2 mM in the light and dark, respectively, similar to those of other C(4) PEPC isoforms. In contrast, in C(3) leaves, transcript expression of both isoforms was weak, with little evidence of diel regulation, and the PEPC proteins showed essentially no indication of phosphorylation. PEPC activity in C(3) leaves was low, light independent and followed Michaelis-Menten kinetics. It was tolerant to malate, with 10-fold higher I(50) values than the PEPC from C(4) leaves. These data suggest that hvpepc4 encodes the C(4) photosynthetic PEPC, and hvpepc3 encodes an anaplerotic form.

Gene Expression Regulation, Plant↗
PubMed1992

Evidence for an arginine residue at the allosteric sites of spinach leaf ADPglucose pyrophosphorylase.

The covalent modification of spinach leaf ADPglucose pyrophosphorylase leads to inactivation of both activator-stimulated and -unstimulated activity. Inactivation can be prevented if either the activator 3PGA or the inhibitor Pi are present during the modification. Pi proved to be more effective at protecting the enzyme from inactivation as it afforded 50% protection at 51 microM compared to 50% protection by 405 microM 3PGA. Partial modification of the enzyme using [14C]-phenylglyoxal leads to a decrease in both Vmax, A0.5 and a decrease in the ability of the 3PGA to stimulate the enzyme's activity. Modification increased the enzyme's susceptibility to inhibition by Pi and completely abolished the cooperative binding of Pi seen in the unmodified enzyme in the presence of 3PGA. Thus, phenylglyoxal appears to interfere, with the normal allosteric regulation of ADPglucose pyrophosphorylase from spinach leaf. Greater than 90% of the enzyme's activity is lost when 7.2 mol [14C]-phenylglyoxal are bound per mole of tetramer and this label is present in both the larger and small subunits. In addition, inactivation appears to involve two different arginine residues having different rates of modification.

Allosteric Site↗