Carboxylation Of acetyl-CoA Research Articles

Regulation of Malonyl-CoA Concentration and Turnover in the Normal Heart

The goal of this study was to test the relationship between malonyl-CoA concentration and its turnover measured in isolated rat hearts perfused with NaH(13)CO(3). This turnover is a direct measurement of the flux of acetyl-CoA carboxylation in the intact heart. It also reflects the rate of malonyl-CoA decarboxylation, i.e. the only known fate of malonyl-CoA in the heart. Conditions were selected to result in stable malonyl-CoA concentrations ranging from 1.5 to 5 nmol.g wet weight-(1). The malonyl-CoA concentration was directly correlated with the turnover of malonyl-CoA, ranging from 0.7 to 4.2 nmol.min(-) (1).g wet weight(-1) (slope = 0.98, r(2) = 0.94). The V(max) activities of acetyl-CoA carboxylase and of malonyl-CoA decarboxylase exceeded the rate of malonyl-CoA turnover by 2 orders of magnitude and did not correlate with either concentration or turnover of malonyl-CoA. However, conditions of perfusion that increased acetyl-CoA supply resulted in higher turnover and concentration, demonstrating that malonyl-CoA turnover is regulated by the supply of acetyl-CoA. The only condition where the activity of malonyl-CoA decarboxylase regulated malonyl-CoA kinetics was when the enzyme was pharmacologically inhibited, resulting in increased malonyl-CoA concentration and decreased turnover. Our data show that, in the absence of enzyme inhibitors, the rate of acetyl-CoA carboxylation is the main determinant of the malonyl-CoA concentration in the heart.

Plant acetyl-CoA carboxylase: structure, biosynthesis, regulation, and gene manipulation for plant breeding.

Acetyl-CoA carboxylase (ACCase) catalyzes the first committed step of fatty acid synthesis, the carboxylation of acetyl-CoA to malonyl-CoA. Two physically distinct types of enzymes are found in nature. Heteromeric ACCase composed of four subunits is usually found in prokaryotes, and homomeric ACCase composed of a single large polypeptide is found in eukaryotes. Most plants have both forms, the heteromeric form in plastids, in which de novo fatty acids are synthesized, and the homomeric form in cytosol. This review focuses on the structure and regulation of plant heteromeric ACCase and its manipulation for plant breeding.

Cyclohexanedione herbicides are inhibitors of rat heart acetyl-CoA carboxylase.

Acetyl CoA carboxylase (ACC) catalyzes the carboxylation of acetyl CoA to form malonyl CoA. In skeletal muscle and heart, malonyl CoA functions to regulate lipid oxidation by inhibition of carnitine palmitoyltransferase-1, an enzyme which controls the entry of long chain fatty acids into mitochondria. We have found that several members of the cyclohexanedione class of herbicides are competitive inhibitors of rat heart ACC. These compounds constitute valuable reagents for drug development and the study of ACCbeta, a validated anti-obesity target.

Multifunctional acetyl-CoA carboxylase 1 is essential for very long chain fatty acid elongation and embryo development in Arabidopsis.

Acetyl-CoA carboxylase (ACCase) catalyses the carboxylation of acetyl-CoA, forming malonyl-CoA, which is used in the plastid for fatty acid synthesis and in the cytosol in various biosynthetic pathways including fatty acid elongation. In Arabidopsis thaliana, ACC1 and ACC2, two genes located in a tandem repeat within a 25-kbp genomic region near the centromere of chromosome 1, encode two multifunctional ACCase isoforms. Both genes, ACC1 and ACC2, appear to be ubiquitously expressed, but little is known about their respective function and importance. Here, we report the isolation and characterisation of two allelic mutants disrupted in the ACC1 gene. Both acc1-1 and acc1-2 mutations are recessive and embryo lethal. Embryo morphogenesis is impaired and both alleles lead to cucumber-like structures lacking in cotyledons, while the shortened hypocotyl and root exhibit a normal radial pattern organisation of the body axis. In this abnormal embryo, the maturation process still occurs. Storage proteins accumulate normally, while triacylglycerides (TAG) are synthesised at a lower concentration than in the wild-type seed. However, these TAG are totally devoid of very long chain fatty acids (VLCFA) and consequently enriched in C18:1, like all lipid fractions analysed in the mutant seed. These data demonstrate, in planta, the role of ACCase 1 in VLCFA elongation. Furthermore, this multifunctional enzyme also plays an unexpected and central function in embryo morphogenesis, especially in apical meristem development.

Surprises and revelations in biochemistry: 1950–2000

Surprises and revelations in biochemistry: 1950–2000

The multisubunit acetyl-CoA carboxylase is strongly associated with the chloroplast envelope through non-ionic interactions to the carboxyltransferase subunits

The committed step for de novo fatty acid biosynthesis is the carboxylation of acetyl-CoA catalyzed by acetyl-CoA carboxylase (ACCase). Plastidial ACCase from most plants is a multisubunit complex composed of multiple copies of four different polypeptides, biotin carboxyl carrier protein (BCCP), biotin carboxylase (BC), and carboxyltransferase (α-CT and β-CT). Immunoblot analyses revealed these four proteins were mostly (69% of total) associated with a 17,000 g insoluble fraction from lysed pea chloroplasts. Under the same conditions only 8% of ribulose-1,5-bisphosphate carboxylase was associated with this insoluble fraction. BCCP and biotin carboxylase BC subunits freely dissociated from 17 kg insoluble fractions under high ionic strength conditions, whereas α-CT and β-CT subunits remained tightly associated. Both CT subunits were highly enriched in envelope versus stroma and thylakoid preparations whereas BC and BCCP subunits were predominantly stromal-localized due to partial dissociation. Rapid solubilization of intact chloroplasts with Triton X-100 followed by centrifugation at 30 kg resulted in a pellet that was up to 8-fold enriched in ACCase activity and 21-fold enriched in BC activity. Triton-insoluble 30 kg pellets were reduced in lipid and chlorophyll content but enriched in chloroplast DNA due to the isolation of nucleoid particles. However, ACCase was not directly associated with nucleoids since enzymatic digestion of DNA or RNA had no effect on the association with Triton-insoluble matter. The amount of Triton-insoluble ACCase was similar in chloroplasts isolated from dark- or light-adaptated leaves suggesting transitory starch granules were also not involved in this association. It is proposed that ACCase is associated with envelope membranes through interactions with an unidentified integral membrane protein.

Indole-3-butyric acid biosynthesis in maize is enhanced by cyclohexanedione herbicides

It has recently been shown that indole-3-butyric acid (IBA) is formed in maize, via acetylation of indoleacetic acid, by a microsomal membrane fraction, ATP and acetyl-CoA. A similarity of this reaction to the carboxylation of acetyl-CoA is proposed and therefore the effects of herbicides specific for the inhibition of acetyl-CoA carboxylase were tested on IBA synthesis. Aryloxyphenoxypropionates and cyclohexanediones inhibited the growth of the maize seedlings as predicted, whereas IBA biosynthesis was promoted after the application of cyclohexanediones. Herbicides with different targets, however, did not increase IBA synthesis. IBA synthetase was not influenced in sethoxydim-tolerant maize seedlings as compared to susceptible plants. It was shown that IBA synthetase from dicotyledonous plants was not affected by sethoxydim application, whereas IBA synthesis in wheat was modulated in the same way as in maize.

Propionyl-CoA carboxylase of Myxococcus xanthus: catalytic properties and function in developing cells.

An acyl-coenzyme A carboxylase that carboxylates acetyl-CoA, butyryl-CoA, propionyl-CoA, and succinyl-CoA was purified from Myxococcus xanthus. Since the enzyme showed maximal rates of carboxylation with propionyl-CoA, the enzyme is thought to be propionyl-CoA carboxylase. The apparent Km values for acetyl-CoA, butyryl-CoA, propionyl-CoA, and succinyl-CoA were found to be 0.2, 0. 2, 0.03, and 1.0 mM, respectively. The native enzyme has a molecular mass of 605-615 kDa and is composed of nonidentical subunits (alpha and beta) with molecular masses of 53 and 56 kDa, respectively. The enzyme showed maximal activity at pH 7.0-7.5 and at 25-30 degrees C, and was affected by variation in concentrations of ATP and Mg2+. During development of M. xanthus, the propionyl-CoA carboxylase activity increased gradually, with maximum activity observed during the sporulation stage. Previous work has shown that a propionyl-CoA-carboxylase-deficient mutant of M. xanthus reduces levels of long-chain fatty acids. These results suggest that the propionyl-CoA carboxylase is also responsible for the carboxylation of acetyl-CoA to malonyl-CoA used for the synthesis of long-chain fatty acids during development.

Genomic Organization of 251 kDa Acetyl-CoA Carboxylase Genes in Arabidopsis: Tandem Gene Duplication has Made Two Differentially Expressed Isozymes

Acetyl-CoA carboxylase (ACCase) catalyzes the carboxylation of acetyl-CoA, forming malonyl-CoA a key intermediate in the biosynthesis of fatty acids and a variety of secondary metabolites. Based upon amino acid sequences conserved among rat, chicken, and E. coli ACCases, PCR-primers were used to amplify a genomic fragment which codes for an ACCase of Arabidopsis. The resulting fragment was used for isolation of genomic and cDNA clones. We have determined the complete cDNA sequence coding for an Arabidopsis ACCase consists of 2,254 amino acids with the molecular mass of 251 kDa. This enzyme contains no recognizable plastid transit-peptide sequence. Therefore, this ACCase is presumably the cytosolic isozyme. Southern analysis indicates that there are two ACCase genes in the Arabidopsis genome. Surprisingly, the results of RFLP analysis and physical mapping of the isolated genomic clones demonstrate that these two genes, acc1 and acc2, are contiguously located within a 25-kbp genomic region near the middle of chromosome 1. Both genes are transcriptionally active, as transcripts from each gene were detected by reverse transcription-PCR analysis using gene-specific primers. The acc1 and acc2 transcripts accumulate in leaves and seedlings but only the acc1 transcript accumulates in developing siliques, unexpectedly. The differences in the expression patterns may be indicative of the differential role of the two genes.

Molecular cloning, characterization, and elicitation of acetyl-CoA carboxylase from alfalfa.

Acetyl-CoA carboxylase [ACCase; acetyl-CoA:carbon-dioxide ligase (ADP-forming), EC 6.4.1.2] catalyzes the ATP-dependent carboxylation of acetyl CoA to produce malonyl CoA. In plants, malonyl CoA is needed for plastid localized fatty acid biosynthesis and for a variety of pathways in the cytoplasm including flavonoid biosynthesis. We have determined the full nucleotide sequence of an ACCase from alfalfa, which appears to represent a cytoplasmic isozyme. Partial cDNAs were isolated from a cDNA library of suspension culture cells that had been elicited for isoflavonoid phytoalexin synthesis. The full-length sequence was obtained by primer extension and amplification of the cDNA with synthetic primers. The sequence codes for a protein of 2257 amino acids with a calculated M(r) of 252,039. The biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase domains, respectively, show approximately 72%, 50%, and 65% sequence similarity to those of animal, diatom, and yeast ACCase sequences. ACCase enzyme activity and transcripts are induced severalfold upon addition of yeast or fungal elicitors to alfalfa cell cultures.

Molecular cloning of two different cDNAs for maize acetyl CoA carboxylase.

Acetyl CoA carboxylase (EC 6.4.1.2) in plants is a chloroplast-localized, biotin-containing enzyme that catalyses the carboxylation of acetyl CoA to malonyl CoA, the first committed step of the fatty acid biosynthesis pathway. Acetyl CoA carboxylase is the target site for the monocotyledon-specific aryloxyphenoxypropionate and cyclohexanedione groups of herbicides. We have purified a herbicide-sensitive acetyl CoA carboxylase from maize leaves to homogeneity (specific activity 7 mumol min-1 mg-1), separating it during the purification from a minor herbicide-resistant acetyl CoA carboxylase. The purified enzyme is a dimer of 230 kDa subunits. Antibodies raised to the purified acetyl CoA carboxylase detected three cross-reacting clones in a maize leaf cDNA expression library, each having an insert of 4-4.5 kb. Restriction analysis and sequencing showed that the cDNAs were derived from two different transcripts. Comparison of the deduced amino acid sequences with those of chicken and yeast acetyl CoA carboxylases confirmed that both types encoded acetyl CoA carboxylase, corresponding to the C-terminal half of the enzyme. The overall identity of the maize and chicken sequences was 37% (58% similarity) but for some shorter regions was much higher. Analysis of six other acetyl CoA carboxylase clones recovered from the maize cDNA library showed four belonged to one type and two to the other. The nucleotide sequence similarity between the two types of cDNA was approximately 95% in the coding region but considerably less in the 3'-untranslated region. Northern blot analysis of maize RNA showed a single band of 8.2-8.5 kb for acetyl CoA carboxylase mRNA. Southern blot hybridisations indicated that there are probably no more than two genes in maize for acetyl CoA carboxylase. The possible significance of two different cDNAs for acetyl CoA carboxylase is discussed.

Studies on the Inhibition of Biotin-Containing Carboxylases by Acetyl-CoA Carboxylase Inhibitors

In higher plants the biosynthetic machinery of de novo fatty acid biosynthesis, measured as [14C]acetate incorporation into fatty acids, is predominantly located in plastids. A key enzyme in this pathway is the biotin-containing acetyl-CoA carboxylase (ACC , EC 6.4.1.2) which catalyzes the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. The ACC from Poaceae is very efficiently blocked by two herbicide classes, the cyclohexane-1,3-diones (e.g. sethoxydim, cycloxydim) and the aryloxyphenoxy-propionic acids (e.g. diclofop, fluazifop). It is shown that within the Poaceae not only different species but also different varieties exist which exhibit an altered sensitivity and tolerance towards both herbicide classes, which points to a mutation of the target enzyme ACC. In purifying the ACC we extended our research to the possible presence of other biotin-containing plant enzymes. In protein preparations from maize, oat, barley, pea and lentil we were able to demonstrate the carboxylation of acetyl-CoA, propionyl-CoA and methylcrotonyl-CoA. The two herbicide classes not only block the ACC, but also the activity of the propionyl-CoA carboxylase (PCC ), whereas the methylcrotonyl- CoA carboxylase (MCC ), a distinct biotin-containing enzyme from mitochondria, is not affected. MCC may play a role in isoprenoid catabolism. Whether PCC is a separate plastid enzyme or only a side activity of ACC is under current investigation. The efficiency of the graminicides in sensitive Poaceae is then not only determined by the inhibition of ACC, malonyl-CoA and fatty acid biosynthesis, but also by the exclusion of the PCC-catalyzed metabolic pathways of the plant cell.

Interaction of acetyl‐CoA fragments with rat liver acetyl‐CoA carboxylase

The interaction of acetyl-CoA fragments with rat liver acetyl-CoA carboxylase has been studied. Dephosphorylated acetyl-CoA did not actually differ from acetyl-CoA in its substrate properties. Non-nucleotide analogues of the substrate, S-acetylpantatheine and it's 4'-phosphate, also possess substrate properties (Vmax = 1.5% and 15% of the maximal rate value of acetyl-CoA carboxylation, respectively). The nucleotide fragment in the acetyl-CoA molecule produces a marked effect on the thermodynamics of the substrate-enzyme interaction, and is apparently involved in activation and appropriate orientation of the acetyl group in the active site. The better substrate properties of S-acetylpantetheine 4'-phosphate and the inhibitory properties of pantetheine 4'-phosphate, compared to the unphosphorylated analogues, evidence an important role of the 5'-beta-phosphate of 3'-phosphorylated ADP residue in acetyl-CoA binding to the enzyme.

13C‐NMR study of acetate assimilation in Thermoproteus neutrophilus

Acetate assimilation into amino acids and the functioning of central biosynthetic pathways in the extremely thermophilic anaerobic archaebacterium Thermoproteus neutrophilus was investigated using 13C NMR as the method for determination of the labelling patterns. Acetate was assimilated via reductive carboxylation of acetyl-CoA to pyruvate and pyruvate conversion to phosphoenolpyruvate which was further carboxylated to oxaloacetate. 2-Oxoglutarate was mainly formed via citrate. However, the labelling patterns of glutamic acid and alanine were in agreement with the concurrent synthesis of about 15% 2-oxoglutarate and 5% pyruvate through the reductive citric acid cycle. A scrambling phenomenon occurring in aspartate and all amino acids derived through oxaloacetate was observed. The labelling patterns of amino acids were in agreement with their standard biosynthetic pathways, with two remarkable exceptions: isoleucine was synthesized via the citramalate pathway and lysine was synthesized via the 2-aminoadipate pathway which has previously been reported only in eukaryotic microorganisms.

Purification and characterization of pyruvate:NADP+ oxidoreductase in Euglena gracilis.

Pyruvate:NADP+ oxidoreductase was homogeneously purified from crude extract of Euglena gracilis. The Mr of the enzyme was estimated to be 309,000 by gel filtration. The enzyme migrated as a single protein band with Mr of 166,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the enzyme consists of two identical polypeptides. The absorption spectrum of the native enzyme exhibited maxima at 278, 380, and 430 nm, and a broad shoulder was observed around 480 nm; the maximum at 430 nm was eliminated by reduction of the enzyme with dithionite. Reduction of the enzyme with pyruvate and CoA and reoxidation with NADP+ were proved from changes of absorption spectra. The enzyme contained 2 molecules of FAD and 8 molecules of iron. It was also indicated that the enzyme was thiamine pyrophosphate-dependent. The enzyme was oxygen-sensitive, and the reaction was affected by the presence of oxygen. Pyruvate was the most active substrate, but the enzyme was slightly active for 2-oxobutyrate, 3-hydroxypyruvate, and oxalacetate, but not for glyoxylate and 2-oxoglutarate. The native electron acceptor was NADP+, whereas NAD+ was completely inactive. Methyl viologen, benzyl viologen, FAD, and FMN were utilized as artificial electron acceptors, whereas spinach and Clostridium ferredoxins were inactive. Pyruvate synthesis by reductive carboxylation of acetyl-CoA with NADPH as the electron donor occurred by the reverse reaction of the enzyme. The enzyme also catalyzed a pyruvate-CO2 exchange reaction and electron-transfer reaction from NADPH to other electron acceptors like methyl viologen. These results indicate that pyruvate:NADP+ oxidoreductase in E. gracilis is clearly distinct from either the pyruvate dehydrogenase multienzyme complex or pyruvate:ferredoxin oxidoreductase.

Carbon assimilation pathways in archaebacteria

The assimilation of cell carbon from CO2 and other compounds has been studied in autotrophic representatives of the archaebactera. The Calvin cycle is not operating in these bacteria. Two different basic pathways for the assimilation of cell carbon from CO2 are found. The methanogenic archaebacteria assimilate CO2 into the central intermediate acetyl CoA via a non-cyclic pathway. The methyl group of acetate is derived from tetrahydropteridin-bound C1-compounds which are also common intermediates in the process of CO2 reduction to methane. The carboxyl group of acetate is formed by CO2 reduction to carbon monoxide. Further assimilation of the activated acetic acid involves 2 to 3 further CO2 fixation reactions and proceeds via the reductive carboxylation of acetyl CoA to pyruvate. The autotrophic sulfur-associated (—“dependent”) archaebacteria appear to use a cyclic reductive carboxylic acid pathway for CO2 assimilation; however, essential enzymes remain to be demonstrated. In the aerobic sulfur oxidizer Sulfolobus the nature of this pathway is still uncertain. In the anaerobic sulfur reducer Thermoproteus a “reductive citric acid cycle” may be operative.

The source of malonyl-CoA in rat heart: The calcium paradox releases acetyl-CoA carboxylase and not propionyl-CoA carboxylase

The formation of malonyl-CoA in rat heart is catalyzed by cytosolic acetyl-CoA carboxylase. The existence of this enzyme in heart is difficult to prove by the abundant occurrence of mitochondrial propionyl-CoA carboxylase, which is also able to catalyze the carboxylation of acetyl-CoA. We used the calcium paradox as a tool to separate cytosolic components from the remaining heart, and found that acetyl-CoA carboxylase activity was preferentially released, like lactate dehydrogenase and carnitine, while propionyl-CoA carboxylase was almost fully retained. Acetyl-CoA carboxylase activity was determined after activation by citrate ion and Mg 2+. The activity decreased to 64% by 48 h of fasting.

Kinetics of citrate-induced activation and polymerization of chick liver acetyl-CoA carboxylase.

The kinetic time course of citrate-induced activation and polymerization (into filaments) of the protomeric form of acetyl-CoA carboxylase were compared to assess the concertedness of the two processes. Rapid-quench techniques were employed to measure the kinetics of activation of the carboxylase-catalyzed reaction by citrate. When enzyme was preincubated with citrate prior to initiating the steady state turnover reaction with acetyl-CoA in the rapid-quench device, the observed rate of carboxylation of acetyl-CoA was apparently linear from the moment of mixing. However, when enzyme was mixed with citrate to initiate the reaction, a lag (t1/2 = 0.7 s) occurred in the approach to steady state carboxylation rate. This lag was independent of enzyme concentration over a 230-fold range and was marginally dependent upon citrate concentration. Over the same range of enzyme concentration, polymerization of carboxylase protomers, as determined by right angle light scattering, was enzyme concentration-dependent in a manner predicted by a single protomer activation step, followed by a rate-limiting dimerization of active protomer and subsequent polymerization. Polymerization is a second order process, with a second order rate constant of 597,000 M-1 s-1. There appear to be two steps that limit polymerization of the inactive carboxylase protomer: a rapid citrate-induced conformational change, which is independent of enzyme concentration and leads to an active protomeric form of the enzyme and the dimerization of the active protomer, which constitutes the first step of polymerization and is enzyme concentration-dependent. Dimerization is the rate-limiting step of acetyl-CoA carboxylase polymerization. On the basis these results, it is concluded that activation of catalysis and the polymerization of carboxylase protomers are not concerted. Furthermore, activation of carboxylation leading to the formation of an active protomer was faster than polymerization under all conditions, and therefore precedes polymerization. It was also shown that the activation constant (Kact) for citrate is altered in a predictable manner by the accumulation of the reaction product, malonyl-CoA, the Kact increasing with malonyl-CoA concentration. Depolymerization of fully polymerized acetyl-CoA carboxylase is caused by malonyl-CoA or ATP.Mg (and HCO3-). Both malonyl-CoA and ATP.Mg (and HCO3-) compete with citrate in the maintenance of a given state of the protomer-polymer equilibrium apparently by carboxylating the enzyme to form enzyme-biotin CO2- which destabilizes the polymeric form.

Propionyl-CoA carboxylase inNocardia mediterranei

The propionyl-CoA carboxylase activity of crude extracts of Nocardia mediterranei fractionated by ammonium sulfate (40–60%) is described. Such an enzyme may play an important role in the biosynthesis of rifamycin as well as in the degradation of various amino acids. The effect of different compounds on the enzymatic reaction have been investigated. Product P8/1-OG and citrate inhibited the enzyme. An activation by the amino acids isoleucine, methionine, threonine and valine was found. The results do not exclude that the same enzyme is responsible for the carboxylation of acetyl-CoA and propionyl-CoA.

Kinetics of activation of acetyl-CoA carboxylase by citrate. Relationship to the rate of polymerization of the enzyme.

The kinetics of citrate-induced activation and polymerization (into filaments) of the 450,000-dalton protomeric form of acetyl-CoA carboxylase were compared to assess the concertedness of the two processes. Rapid-quench techniques were employed to measure the time course of activation by citrate of the carboxylase-catalyzed reaction. When enzyme was preincubated with citrate prior to initiating the steady state turnover reaction with acetyl-CoA in the rapid-quench device, the observed rate of carboxylation of acetyl-CoA was apparently linear from the moment of mixing. However, when enzyme was mixed with citrate to initiate the reaction, a lag (t1/2 = 0.7 s) occurred in the approach to steady state carboxylation rate. This lag was independent of enzyme concentration over a 230-fold range and was marginally dependent upon citrate concentration. Over the same range of enzyme concentration, polymerization of carboxylase protomers, as determined by right angle light scattering, was enzyme concentration-dependent in a manner predicted by a single protomer activation step, followed by a rate-limiting dimerization of active protomer and subsequent polymerization. Based on these results, it is concluded that activation of catalysis and the polymerization of carboxylase protomers are not concerted. Furthermore, activation of carboxylation leading to the formation of an active protomer was faster than polymerization under all conditions, and therefore precedes polymerization. It was also shown that the activation constant (Kact) for citrate is altered in a predictable manner by the accumulation of the reaction product, malonyl-CoA, the Kact increasing with increasing malonyl-CoA concentration. Additional evidence is presented indicating that this change in Kact was not caused by autophosphorylation of the enzyme under these conditions and that phosphorylation does not affect the mechanism of activation elucidated above.