Characterization of point mutations in patients with pyruvate dehydrogenase deficiency: role of methionine-181, proline-188, and arginine-349 in the alpha subunit.

Human pyruvate dehydrogenase (E1), a heterotetramer (alpha2beta2), is the first component of the pyruvate dehydrogenase complex (PDC). E1 catalyzes the thiamin pyrophosphate (TPP)-dependent decarboxylation of pyruvate and the reductive acetylation of the dihydrolipoamide acetyltransferase component. Site-directed mutagenesis was employed to recreate three point mutations in the alpha subunit identified in E1-deficient patients, M181V, R349H, and P188L (P188A mutant E1 was used because of the very low level of expression of P188L), to investigate the functional roles of these three amino acid residues. P188A mutant E1 was much less thermostable than the wild-type E1. The kcats of M181V and P188A mutant E1s determined in the PDC reaction were 38 and 24% of that of the wild-type enzyme, respectively. The apparent Km for TPP for M181V increased significantly (approx 250-fold when determined in the PDC assay), while the apparent Km for pyruvate increased by only about 3-fold. In contrast, P188A had similar Kms for the coenzyme and the substrate as the wild-type. Km values for R349H were not determined due to the extremely low activity of this mutant (1.2% of the wild-type E1-specific activity measured in the PDC assay). Wild-type E1 displayed a lag phase in the progress curve of the PDC reaction measured in the presence of low TPP concentrations (below 1 microM) only. All mutants had a lag phase that was not eliminated even at very high TPP concentrations, suggesting modifications in the conformation of the active site. Kinetic analysis indicated thiamin 2-thiothiazolone pyrophosphate (ThTTPP) to be an intermediate analog for wild-type human E1. M181V required a higher concentration of ThTTPP for inactivation than the wild-type and P188A E1s. The results of circular dichroism spectropolarimetry in the far UV region indicated that there were no major changes in the secondary structure of M181V, P188A, and R349H E1s. These mutant enzymes exhibited negative dichroic spectra at about 330 nm only in the presence of high TPP concentrations. This study suggests that arginine-349 is critical for E1's activity, methionine-181 is involved in the binding of TPP, and proline-188 is necessary for structural integrity of E1.     

The elementary reactions of the pig heart pyruvate dehydrogenase complex. A study of the inhibition by phosphorylation.

1. A method was devised for preparing pig heart pyruvate dehydrogenase free of thiamin pyrophosphate (TPP), permitting studies of the binding of [35S]TPP to pyruvate dehydrogenase and pyruvate dehydrogenase phosphate. The Kd of TPP for pyruvate dehydrogenase was in the range 6.2-8.2 muM, whereas that for pyruvate dehydrogenase phosphate was approximately 15 muM; both forms of the complex contained about the same total number of binding sites (500 pmol/unit of enzyme). EDTA completely inhibited binding of TPP; sodium pyrophosphate, adenylyl imidodiphosphate and GTP, which are inhibitors (competitive with TPP) of the overall pyruvate dehydrogenase reaction, did not appreciably affect TPP binding. 2. Initial-velocity patterns of the overall pyruvate dehydrogenase reaction obtained with varying TPP, CoA and NAD+ concentrations at a fixed pyruvate concentration were consistent with a sequential three-site Ping Pong mechanism; in the presence of oxaloacetate and citrate synthase to remove acetyl-CoA (an inhibitor of the overall reaction) the values of Km for NAD+ and CoA were 53+/- 5 muM and 1.9+/-0.2 muM respectively. Initial-velocity patterns observed with varying TPP concentrations at various fixed concentrations of pyruvate were indicative of either a compulsory order of addition of substrates to form a ternary complex (pyruvate-Enz-TPP) or a random-sequence mechanism in which interconversion of ternary intermediates is rate-limiting; values of Km for pyruvate and TPP were 25+/-4 muM and 50+/-10 nM respectively. The Kia-TPP (the dissociation constant for Enz-TPP complex calculated from kinetic plots) was close to the value of Kd-TPP (determined by direct binding studies). 3. Inhibition of the overall pyruvate dehydrogenase reaction by pyrophosphate was mixed non-competitive versus pyruvate and competitive versus TPP; however, pyrophosphate did not alter the calculated value for Kia-TPP, consistent with the lack of effect of pyrophosphate on the Kd for TPP. 4. Pyruvate dehydrogenase catalysed a TPP-dependent production of 14CO2 from [1-14C]pyruvate in the absence of NAD+ and CoA at approximately 0.35% of the overall reaction rate; this was substantially inhibited by phosphorylation of the enzyme both in the presence and absence of acetaldehyde (which stimulates the rate of 14CO2 production two- or three-fold). 5. Pyruvate dehydrogenase catalysed a partial back-reaction in the presence of TPP, acetyl-CoA and NADH. The Km for TPP was 4.1+/-0.5 muM. The partial back-reaction was stimulated by acetaldehyde, inhibited by pyrophosphate and abolished by phosphorylation. 6. Formation of enzyme-bound [14C]acetylhydrolipoate from [3-14C]pyruvate but not from [1-14C]acetyl-CoA was inhibited by phosphorylation. Phosphorylation also substantially inhibited the transfer of [14C]acetyl groups from enzyme-bound [14C]acetylhydrolipoate to TPP in the presence of NADH. 7...     

Kinetic characterization of the pyruvate and oxoglutarate dehydrogenase complexes from human heart

The Michaelis constant values for the highly purified pyruvate dehydrogenase complex (PDC) from human heart are 25, 13 and 50 microM for pyruvate, CoA and NAD, respectively. Acetyl-CoA produces a competitive inhibition of PDC (Ki = 35 microM) with respect to CoA, whereas NADH produces the same type of inhibition with respect to NAD (Ki = 36 microM). The oxoglutarate dehydrogenase complex (OGDC) from human heart has active sites with two different affinities for 2-oxoglutarate ([S]0.5 of 30 and 120 microM). ADP (1 mM) decreases the [S]0.5 values by a half. The inhibition of OGDC (Ki = 81 microM) by succinyl-CoA is of a competitive type with respect to CoA (Km = 2.5 microM), whereas that of NADH (Ki = 25 microM) is of a mixed type with respect to NAD (Km = 170 microM).     

Bovine heart pyruvate dehydrogenase kinase stimulation by alpha-ketoisovalerate

Purified bovine heart pyruvate dehydrogenase complex was used to investigate the effects of monovalent cations and alpha-ketoisovalerate on pyruvate dehydrogenase (PDH) kinase inhibition by thiamin pyrophosphate. Initial velocity patterns for thiamin pyrophosphate inhibition were consistent with hyperbolic non-competitive or hyperbolic uncompetitive inhibition at various K+ concentrations between 0 and 120 mM. The Kis, Kid, and Kin for thiamin pyrophosphate were in the range of 0.009 to 5.1 microM over the range of K+ concentrations tested. In the absence of K+, 1 mM alpha-ketoisovalerate had no effect on PDH kinase inhibition by thiamin pyrophosphate, whereas in the presence of 20 mM K+, alpha-ketoisovalerate stimulated PDH kinase activity almost 2-fold over the range of 0-80 microM thiamin pyrophosphate. Half-maximal stimulation by alpha-ketoisovalerate occurred at about 200 microM in the presence of 100 microM thiamin pyrophosphate and 20 mM K+. Similar but less extensive changes occurred in the presence of 100 microM thiamin pyrophosphate and 1 mM NH4+. Initial velocity patterns for PDH kinase inhibition by thiamin pyrophosphate in the presence of 2 mM alpha-ketoisovalerate were mixed noncompetitive, but alpha-ketoisovalerate increased the Vm and Km for adenosine 5'-triphosphate in the presence of inhibitor. In the presence of thiamin pyrophosphate, PDH kinase remained stimulated after chromatography on Sephadex G-25 to remove alpha-ketoisovalerate. The results indicate that acylation of pyruvate dehydrogenase complex by alpha-ketoisovalerate results in PDH kinase stimulation but only in the presence of monovalent cations and thiamin pyrophosphate.     

Elementary steps in the reaction of the pyruvate dehydrogenase complex from pig heart. Kinetics of thiamine diphosphate binding to the complex

In the progress curve of the reaction of the pyruvate dehydrogenase complex, a lag phase was observed when the concentration of thiamin diphosphate was lower than usual (about 0.2-1 mM) in the enzyme assay. The length of the lag phase was dependent on thiamin diphosphate concentration, ranging from 0.2 min to 2 min as the thiamin diphosphate concentration varied from 800 nM to 22 nM. The lag phase was also observed in the elementary steps catalyzed by the pyruvate dehydrogenase component. A Km value of 107 nM was found for thiamin diphosphate with respect to the steady-state reaction rate following the lag phase. The pre-steady-state kinetic data indicate that the resulting lag phase was the consequence of a slow holoenzyme formation from apoenzyme and thiamin diphosphate. The thiamin diphosphate can bind to the pyruvate dehydrogenase complex in the absence of pyruvate, but the presence of 2 mM pyruvate increases the rate constant of binding from 1.4 X 10(4) M-1 S-1 to 1.3 X 10(5) M-1 S-1 and decreases the rate constant of dissociation from 2.3 X 10(-2) S-1 to 4.1 X 10(-3) S-1. On the other hand, the effect of pyruvate on the thiamin diphosphate binding revealed the existence of a thiamin-diphosphate-independent pyruvate-binding site in the pyruvate dehydrogenase complex. Direct evidence was also obtained with fluorescence techniques for the existence of this binding site and the dissociation constant of pyruvate was found to be 0.38 mM. On the basis of these data we have proposed a random mechanism for the binding of pyruvate and thiamin diphosphate to the complex. Binding of substrates to the enzyme complex caused an increase in the fluorescence of the dansylaziridine-labelled pyruvate dehydrogenase complex, showing that binding of substrates to the complex is accompanied by structural changes.     

Properties of the thiamin triphosphate-synthesizing activity catalyzed by adenylate kinase (isoenzyme 1)

Adenylate kinase isozyme 1 (AK1) catalyzes thiamin triphosphate (TTP) formation from thiamin diphosphate (TDP) and ADP. The properties of the TTP-synthesizing activity of purified AK1 from porcine skeletal muscle were studied. The activity was found to require TDP, ADP, and Mg2+, and ATP was only 14.4% as active as ADP. Thiamin monophosphate (TMP) and thiamin were not utilized as substrates. ADP was specific as a phosphate donor; and CDP, UDP, and GDP supported TTP formation at rates less than 1% of that with ADP. Optimal pH and temperature for the TTP-synthesizing activity were 10.0 and 37 degrees C, respectively. The activity showed saturation kinetics for both substrates, and the Km values for TDP and ADP were calculated to be 0.83 mM and 43 microM, respectively. The enzyme catalyzed the reverse reaction (TTP + AMP----TDP + ADP) and stoichiometry between TTP and TDP was demonstrated in the forward and reverse reactions.     

A synthesis of acylphosphonic acids and of 1-aminoalkylphosphonic acids: the action of pyruvate dehydrogenase and lactate dehydrogenase on acetylphosphonic acid.

Acylphosphonic acids, R-CO-PO(OH)2, have been synthesized by the steps [formula: see text] of which the last is new and provides a mild method for de-esterifying acylphosphonic acids. Their reductive amination gives a simple way of making 1-aminoalkylphosphonic acids. Acetylphosphonic acid inhibited NAD+ reduction by pyruvate with the pyruvate dehydrogenases from Escherichia coli and Bacillus stearothermophilus. The inhibition was competitive with pyruvate, with Ki of 6 microM for the E. coli enzyme (pyruvate Km 0.5 mM) and one of 0.4 mM of the B. stearothermophilus enzyme (pyruvate Km 0.1 mM). Acetylphosphonate and its monomethyl ester are substates for pig heart lactate dehydrogenase, with Km values of 15 mM and 10 mM respectively (pyruvate Km 0.05 mM) and specificity constants one thousandth that for pyruvate.     

Regulation of heart muscle pyruvate dehydrogenase kinase

1. The activity of pig heart pyruvate dehydrogenase kinase was assayed by the incorporation of [(32)P]phosphate from [gamma-(32)P]ATP into the dehydrogenase complex. There was a very close correlation between this incorporation and the loss of pyruvate dehydrogenase activity with all preparations studied. 2. Nucleoside triphosphates other than ATP (at 100mum) and cyclic 3':5'-nucleotides (at 10mum) had no significant effect on kinase activity. 3. The K(m) for thiamin pyrophosphate in the pyruvate dehydrogenase reaction was 0.76mum. Sodium pyrophosphate, adenylyl imidodiphosphate, ADP and GTP were competitive inhibitors against thiamin pyrophosphate in the dehydrogenase reaction. 4. The K(m) for ATP of the intrinsic kinase assayed in three preparations of pig heart pyruvate dehydrogenase was in the range 13.9-25.4mum. Inhibition by ADP and adenylyl imidodiphosphate was predominantly competitive, but there was nevertheless a definite non-competitive element. Thiamin pyrophosphate and sodium pyrophosphate were uncompetitive inhibitors against ATP. It is suggested that ADP and adenylyl imidodiphosphate inhibit the kinase mainly by binding to the ATP site and that the adenosine moiety may be involved in this binding. It is suggested that thiamin pyrophosphate, sodium pyrophosphate, adenylyl imidodiphosphate and ADP may inhibit the kinase by binding through pyrophosphate or imidodiphosphate moieties at some site other than the ATP site. It is not known whether this is the coenzyme-binding site in the pyruvate dehydrogenase reaction. 5. The K(m) for pyruvate in the pyruvate dehydrogenase reaction was 35.5mum. 2-Oxobutyrate and 3-hydroxypyruvate but not glyoxylate were also substrates; all three compounds inhibited pyruvate oxidation. 6. In preparations of pig heart pyruvate dehydrogenase free of thiamin pyrophosphate, pyruvate inhibited the kinase reaction at all concentrations in the range 25-500mum. The inhibition was uncompetitive. In the presence of thiamin pyrophosphate (endogenous or added at 2 or 10mum) the kinase activity was enhanced by low concentrations of pyruvate (25-100mum) and inhibited by a high concentration (500mum). Activation of the kinase reaction was not seen when sodium pyrophosphate was substituted for thiamin pyrophosphate. 7. Under the conditions of the kinase assay, pig heart pyruvate dehydrogenase forms (14)CO(2) from [1-(14)C]pyruvate in the presence of thiamin pyrophosphate. Previous work suggests that the products may include acetoin. Acetoin activated the kinase reaction in the presence of thiamin pyrophosphate but not with sodium pyrophosphate. It is suggested that acetoin formation may contribute to activation of the kinase reaction by low pyruvate concentrations in the presence of thiamin pyrophosphate. 8. Pyruvate effected the conversion of pyruvate dehydrogenase phosphate into pyruvate dehydrogenase in rat heart mitochondria incubated with 5mm-2-oxoglutarate and 0.5mm-l-malate as respiratory substrates. It is suggested that this effect of pyruvate is due to inhibition of the pyruvate dehydrogenase kinase reaction in the mitochondrion. 9. Pyruvate dehydrogenase kinase activity was inhibited by high concentrations of Mg(2+) (15mm) and by Ca(2+) (10nm-10mum) at low Mg(2+) (0.15mm) but not at high Mg(2+) (15mm).     

Pyruvate Decarboxylase from Zea mays L. : 2. Examination of Hysteretic Kinetics

A significant lag phase was observed in the accumulation of product for the reaction catalyzed by pyruvate decarboxylase (PDC) purified from mature maize kernels. The effects of pH, pyruvate, potassium chloride, PDC concentration, and Mg²⁺-thiamine pyrophosphate upon this lag and upon the observed cooperativity were investigated. PDC preincubated with Mg²⁺-thiamine pyrophosphate for six days had Michaelis-Menten kinetics, a Hill number of 1, and no apparent lag phase. The degree of saturation of PDC with Mg²⁺-thiamine pyrophosphate appears to have a central role in controlling the lag phase and the degree of cooperativity.     

Existence in animal tissues of adenosine triphosphate thiamin diphosphate phosphotransferase [EC 2.7.4.15]

An enzyme which catalyzes the synthesis of thiamin triphosphate from thiamin diphosphate (TDP), thiamindiphosphate kinase (ATP:thiamin diphosphate phosphotransferase) [EC 2.7.4.15], was detected in animal tissues. The enzyme was partially purified (150-fold) from the cytosol fraction of guinea pig brain. The enzyme reaction required free (not protein-bound) TDP, ATP, Mg2+, and a cofactor, which is a low molecular weight and heat-stable compound. The enzyme activity was optimal at pH 11 and at 25 degrees C. A stoichiometric transfer of 32P from [gamma-32P]ATP to TDP was demonstrated. Km values for TDP and ATP were calculated to be 1.1 mM and 10 microM, respectively, and Vmax was 868 nmol/mg of protein/hr. The enzyme was found solely in the cytosol fraction of guinea pig brain and was also detectable in the skeletal muscle and heart. These results provide strong evidence for the existence of TDP kinase in animal tissues.     

Mitochondrial NADP-dependent malic enzyme of cod heart. Rate of forward and reverse reaction

The mitochondrial NADP-dependent malic enzyme (EC 1.1.1.40) was purified about 300-fold from cod Gadus morhua heart to a specific activity of 48 units (mumol/min)/mg at 30 degrees C. The possibility of the reductive carboxylation of pyruvate to malate was studied by determination of the respective enzyme properties. The reverse reaction was found to proceed at about five times the velocity of the forward rate at a pH 6.5. The Km values determined at pH 7.0 for pyruvate, NADPH and bicarbonate in the carboxylation reaction were 4.1 mM, 15 microM and 13.5 mM, respectively. The Km values for malate, NADP and Mn2+ in the decarboxylation reaction were 0.1 mM, 25 microM and 5 microM, respectively. The enzyme showed substrate inhibition at high malate concentrations for the oxidative decarboxylation reaction at pH 7.0. Malate inhibition suggests a possible modulation of cod heart mitochondrial NADP-malic enzyme by its own substrate. High NADP-dependent malic enzyme activity found in mitochondria from cod heart supports the possibility of malate formation under conditions facilitating carboxylation of pyruvate.     

Regulatory properties of the pyruvate dehydrogenase complex from Escherichia coli. Studies on the thiamin diphosphate-dependent lag phase

The pyruvate dehydrogenase complex from Escherichia coli shows an appreciable lag phase (tau) of some minutes when its overall reaction rate was tested with very limiting amounts of thiamin diphosphate. tau depends on the concentration of thiamin diphosphate in a nonlinear fashion. Sodium diphosphate, a competitive inhibitor with respect to thiamin diphosphate (Ki = 5.2 . 10(-4) M) prolongs the lag, while the strongly binding transition state analog thiamin thiazolone diphosphate has no effect. tau is independent of the enzyme concentration, thus no dissociation-association step is involved. Incubation of the pyruvate dehydrogenase complex with thiamin diphosphate, Mg2+, and pyruvate leads to a shortening of the lag phase, as well as to a decrease of the intrinsic tryptophan fluorescence in a time-dependent process, which evinces the same characteristics as tau. Dependence of pyruvate, as well as of the substrate analog methylacetylphosphonate, can be established by measurements of fluorescence quenching, thus ruling out an essential role of hydroxyethyl thiamin diphosphate in the process reflected by the lag phase. The results demonstrate that the lag phase is induced after the binding of both thiamin diphosphate . Mg2+ and pyruvate to the catalytic site to form a ternary enzyme complex, which undergoes subsequently a slow conformational change to an active enzyme form. This change is confined to single subunits, and no interactions between neighboring monomers could be observed. A model is proposed to describe the mechanism represented by the lag phase.     

Some properties of the reaction catalyzed by protein kinase bound to cardiac sarcolemma

A highly purified sarcolemmal fraction from rat heart consisted of closed inside-out oriented vesicles and possessed high activities of Na+, K+-ATPase, adenylate cyclase and creatine kinase. Contaminations of sarcolemmal preparation by other membranous fractions were practically absent. This sarcolemmal fraction contained protein kinase tightly bound to the membrane. Substrates of the phosphorylation reaction catalyzed by this protein kinase were either endogenous sarcolemmal protein (proteins) with molecular weight of 11500 or exogenous protein--histone, type II. Phosphorylation of the endogenous but not of the exogenous substrate was completely independent of cyclic AMP. A kinetic analysis of the sarcolemmal protein kinase reaction with Mg[gamma-32P]ATP and histone as substrates revealed that the kinetic mechanism of this reaction is characterized by the following kinetic parameters: Km (Mg-ATP) = 12.1 microM; Km (histone) = 0.47 mg/ml; Ki (Mg-ADP) = 15.6 microM. A comparison of experimental results to literary data allows to suggest that the sarcolemmal enzyme is virtually soluble protein kinase tightly bound to the sarcolemma.     

Effects of alpha-ketoisovalerate on bovine heart pyruvate dehydrogenase complex and pyruvate dehydrogenase kinase

The regulatory effects of alpha-ketoisovalerate on purified bovine heart pyruvate dehydrogenase complex and endogenous pyruvate dehydrogenase kinase were investigated. Incubation of pyruvate dehydrogenase complex with 0.125 to 10 mM alpha-ketoisovalerate caused an initial lag in enzymatic activity, followed by a more linear but inhibited rate of NADH production. Incubation with 0.0125 or 0.05 mM alpha-ketoisovalerate caused pyruvate dehydrogenase inhibition, but did not cause the initial lag in pyruvate dehydrogenase activity. Gel electrophoresis and fluorography demonstrated the incorporation of acyl groups from alpha-keto[2-14C]isovalerate into the dihydrolipoyl transacetylase component of the enzyme complex. Acylation was prevented by pyruvate and by arsenite plus NADH. Endogenous pyruvate dehydrogenase kinase activity was stimulated specifically by K+, in contrast to previous reports, and kinase stimulation by K+ correlated with pyruvate dehydrogenase inactivation. Maximum kinase activity in the presence of K+ was inhibited 62% by 0.1 mM thiamin pyrophosphate, but was inhibited only 27% in the presence of 0.1 mM thiamin pyrophosphate and 0.1 mM alpha-ketoisovalerate. Pyruvate did not affect kinase inhibition by thiamin pyrophosphate at either 0.05 or 2 mM. The present study demonstrates that alpha-ketoisovalerate acylates heart pyruvate dehydrogenase complex and suggests that acylation prevents thiamin pyrophosphate-mediated kinase inhibition.     

Overexpression,purification, and characterization of the periplasmic space thiamin-binding protein of the thiamin traffic ATPase in Escherichia coli

Thiamin (Vitamin B(1)) transport in Escherichia coli occurs by the superfamily of traffic ATPases in which the initial receptor is the periplasmic binding protein. We have cloned the periplasmic thiamin-binding protein (TBP) of the E. coli periplasmic thiamin transport system and purified the overexpressed protein to apparent homogeneity. A subsequent biochemical characterization demonstrates that TBP is a 34.205kDa monomer. TBP also contains one tightly bound thiamin species [thiamin, thiamin monophosphate (TMP), or thiamin diphosphate (TDP)] per monomer (K(D)=0.8 microM) when isolated under conditions that would remove any loosely bound ligands. We also demonstrate that thiamin is readily exchangeable in the presence of exogenous thiamin with a k(off)=0.12s(-1). The biochemical characteristics of the overexpressed, plasmid-derived TBP are indistinguishable from those determined for endogenous TBP purified from E. coli. The overexpression and purification of TBP that we present here allows the rapid isolation of large amounts of pure protein that are required for further mechanistic and structural studies and demonstrates a vast improvement over previously reported purifications.     

Reaction mechanism for mammalian pyruvate dehydrogenase using natural lipoyl domain substrates

The pyruvate dehydrogenase (E1) component of the pyruvate dehydrogenase complex (PDC) catalyzes a two-step reaction. Recombinant production of substrate amounts of the lipoyl domains of the dihydrolipoyl transacetylase (E2) component of the mammalian PDC allowed kinetic characterization of the rapid physiological reaction catalyzed by E1. Using either the N-terminal (L1) or the internal (L2) lipoyl domain of E2 as a substrate, analyses of steady state kinetic data support a ping pong mechanism. Using standard E1 preparations, Michaelis constants (Km) were 52 +/- 14 microM for L1 and 24.8 +/- 3.8 microM for pyruvate and k(cat) was 26.3 s(-1). With less common, higher activity preparations of E1, the Km values were > or =160 microM for L1 and > or =35 microM for pyruvate and k(cat) was > or =70 s(-1). Similar results were found with the L2 domain. The best synthetic lipoylated-peptide (L2 residues 163-177) was a much poorer substrate (Km > or =15 mM, k(cat) approximately equals 5 s(-1); k(cat)/Km decreased >1,500-fold) than L1 or L2, but a far better substrate in the E1 reaction than free lipoamide (k(cat)/Km increased >500-fold). Each lipoate source was an effective substrate in the dihydrolipoyl dehydrogenase (E3) reaction, but E3 had a lower Km for the L2 domain than for lipoamide or the lipoylated peptides. In contrast to measurements with slow E1 model reactions that use artificial acceptors, we confirmed that the natural E1 reaction, using lipoyl domain acceptors, was completely inhibited (>99%) by phosphorylation of E1 and the phosphorylation strongly inhibited the reverse of the second step catalyzed by E1. The mechanisms by which phosphorylation interferes with E1 activity is interpreted based on accrued results and the location of phosphorylation sites mapped onto the 3-D structure of related alpha-keto acid dehydrogenases.     

Regulation of pea mitochondrial pyruvate dehydrogenase complex activity: inhibition of ATP-dependent inactivation

In contrast to the pyruvate dehydrogenase complex (PDC) from animal mitochondria, our in situ and in vitro studies indicate that the ATP:ADP ratio has little or no effect in regulating the mitochondrial pyruvate dehydrogenase complex from green pea seedlings. Pyruvate was a competitive inhibitor of ATP-dependent inactivation (Ki = 59 microM), while the PDC had a Km for pyruvate of microM. Thiamine pyrophosphate, the coenzyme for the pyruvate dehydrogenase (PDH) component of the complex, did not inhibit ATP-dependent inactivation when used alone but it enhanced inhibition by pyruvate. As such, thiamine pyrophosphate was a competitive inhibitor (Ki = 130 nM) of ATP-dependent inactivation. A model is proposed for the pyruvate plus thiamine pyrophosphate inhibition of ATP-dependent inactivation of the pyruvate dehydrogenase complex in which pyruvate exerts its inhibition of inactivation by altering or protecting the protein substrate from phosphorylation and not by directly inhibiting PDH kinase.     

Relative reactivity of thiamine monophosphate and thiamine diphosphate upon interaction with alkaline phosphatase

Reactivity of thiamin monophosphate (TMP) as calf intestinal alkaline phosphatase substrate in model transformations is lower comparing with thiamin diphosphate (TDP) reactivity. Under these conditions alkaline phosphatase catalyzes TDP, ADP and AMP hydrolysis approximately at same rate. It was shown that TDP competes with p-nitrophenyl phosphate more effectively than TMP for the binding in the active site. At pH 8.5 and 30 degrees C Km values are as follows: (5.2 +/- 1.6) x 10(-3) M for TMP and (3.0 +/- 0.8) x 10(-4) M for TDP. Under the same conditions the Vmax/Km value for TDP hydrolysis is 53 times higher than the one for corresponding reaction of TMP. It was suggested that positively charged thiazolium ion of TMP interacts with the nearest environment at the active center and by this way reduces enzyme activity.