Structural and functional analysis of pyruvate kinase from Corynebacterium glutamicum.

Pyruvate kinase activity is an important element in the flux control of the intermediate metabolism. The purified enzyme from Corynebacterium glutamicum demonstrated a marked sigmoidal dependence of the initial rate on the phosphoenolpyruvate concentration. In the presence of the negative allosteric effector ATP, the phosphoenolpyruvate concentration at the half-maximum rate (S0.5) increased from 1.2 to 2.8 mM, and cooperation, as expressed by the Hill coefficient, increased from 2.0 to 3.2. AMP promoted opposite effects: the S0.5 was decreased to 0.4 mM, and the enzyme exhibited almost no cooperation. The maximum reaction rate was 702 U/mg, which corresponded to an apparent kcat of 2,540 s-1. The enzyme was not influenced by fructose-1,6-diphosphate and used Mn2+ or Co2+ as cations. Sequence determination of the C. glutamicum pyk gene revealed an open reading frame coding for a polypeptide of 475 amino acids. From this information and the molecular mass of the native protein, it follows that the pyruvate kinase is a tetramer of 236 kDa. Comparison of the deduced polypeptide sequence with the sequences of other bacterial pyruvate kinases showed 39 to 44% homology, with some regions being very strongly conserved.     

Effect of methylguanidine and guanidinosuccinic acid on pyruvate kinase activity in human red cells

An effect of methylguanidine and guanidinosuccinic acid on pyruvate kinase activity in human red cells was determined in vitro following a 3-hour incubation at 37 degrees C. The obtained results have shown that methylguanidine in the concentration of 1.8 x 10(-5) M/l inhibits pyruvate kinase activity by 20.8%. Pyruvate kinase activity was statistically significantly inhibited on addition of methylguanidine in the concentration of 5.4 x 10(-5) M/l whereas higher concentrations have no such an effect Guanidinesuccinic acid exerted similar but weaker effect on the activity of pyruvate kinase in human red cells. Mixture of methylguanidine (5.4 x 10(-5) m/l) and guanidinesuccinic acid (2.8 x 10(-5) M/l) does not affect pyruvate kinase activity in normal human red cells under identical experimental conditions.     

Genetic analysis of the human thymidine kinase gene promoter.

The promoter of the human thymidine kinase gene was defined by DNA sequence and genetic analyses. Mutant plasmids with deletions extending into the promoter region from both the 5' and 3' directions were constructed. The mutants were tested in a gene transfer system for the ability to transform TK- cells to the TK+ phenotype. This analysis delimited the functional promoter to within an 83-base-pair region upstream of the mRNA cap site. This region contains sequences common to other eucaryotic promoters including G X C-rich hexanucleotides, a CAAT box, and an A X T-rich region. The CAAT box is in an inverted orientation and is part of a 9-base-pair sequence repeated twice in the promoter region. Comparison of the genomic sequence with the cDNA sequence defined the first exon of the thymidine kinase gene.     

Purification and characterization of human muscle pyruvate kinase.

The M1 isozyme of pyruvate kinase has been purified from human psoas muscle in a seven-step procedure. Fractionation by ammonium sulfate precipitation, heat treatment, acetone precipitation, diethylaminoethyl cellulose batchwise treatment followed by chromatography on carboxymethyl cellulose and Sephadex G-200 gave a product with a specific activity of 383 U/mg representing a 294-fold purification with a yield of 11%. The product formed orthorhombic crystals and was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate, sedimentation velocity, sedimentation equilibrium, and immunodiffusion. The purified enzyme has a molecular weight of 240700 and has a sedimentation coefficient (S20,W) of 10.04S. It contains four subunits with identical molecular weights of 61000. No free N-terminal amino acids could be detected. Antibody prepared against the purified human M1 isozyme does not cross-react by immunodiffusion or enzyme inactivation with the human erythrocyte isozyme and in the reverse experiment antibody prepared against human erythrocyte pyruvate kinase does not cross-react with the purified M1 isozyme. The amino acid composition of the M1 isozyme is presented.     

Structural determinants for cross-talk between pyruvate dehydrogenase kinase 3 and lipoyl domain 2 of the human pyruvate dehydrogenase complex

Pyruvate dehydrogenase kinase isoforms (PDK1-4) are the molecular switch that down-regulates activity of the human pyruvate dehydrogenase complex through reversible phosphorylation. We showed previously that binding of the lipoyl domain 2 (L2) of the pyruvate dehydrogenase complex to PDK3 induces a "cross-tail" conformation in PDK3, resulting in an opening of the active site cleft and the stimulation of kinase activity. In the present study, we report that alanine substitutions of Leu-140, Glu-170, and Glu-179 in L2 markedly reduce binding affinities of these L2 mutants for PDK3. Unlike wildtype L2, binding of these L2 mutants to PDK3 does not preferentially reduce the affinity of PDK3 for ADP over ATP. The inefficient removal of product inhibition associated with ADP accounts for the decreased stimulation of PDK3 activity by these L2 variants. Serial truncations of the PDK3 C-terminal tail region either impede or abolish the binding of wild-type L2 to the PDK3 mutants, resulting in the reduction or absence of L2-enhanced kinase activity. Alanine substitutions of residues Leu-27, Phe-32, Phe-35, and Phe-48 in the lipoyl-binding pocket of PDK3 similarly nullify L2 binding and L2-stimulated PDK3 activity. Our results indicate that the above residues in L2 and residues in the C-terminal region and the lipoyl-binding pocket of PDK3 are critical determinants for the cross-talk between L2 and PDK3, which up-regulates PDK3 activity.     

Coupling of glucose response element from L-type pyruvate kinase and G6Pase promoter enhances glucose responsive activity in hepatoma cells

Type 1 diabetes results from the autoimmune destruction of pancreatic β-cells, which leads to severe insulin deficiency. Insulin gene therapy provides an attractive approach to cure diabetes. The critical factor for insulin gene therapy in surrogate cells is to select an appropriate site for insulin expression and a tissue-specific promoter that is responsive to both physiological glucose and insulin concentrations. A novel chimeric promoter, (GIRE)n-G6Pase, consisting of a 1.6 kb glucose 6-phosphatase (G6Pase) promoter and a segment of the regulatory element derived from the L-type pyruvate kinase (L-PK) promoter, was designed to provide strong and tight control of insulin expression in liver. One or three copies of GIRE were linked to the G6Pase promoter, which showed a stronger promoter activity than the G6Pase promoter alone. The chimeric promoter was inhibited by insulin in a dosage-dependent manner and activated by glucose, two features essential for glucose metabolism. The promoter activity is conserved between species and highly specific for liver cells. The construction of a chimeric promoter with stronger and more sensitive responsive activity to glucose and insulin in liver cells could further advance studies in insulin gene therapy. Mr. James Chong was a senior student in the Department of Cell and Molecular Biology, Tulane University. His independent study project was partially overlapped with this study.     

Rapid upregulation of pyruvate dehydrogenase kinase activity in human skeletal muscle during prolonged exercise.

Prolonged moderate-intensity exercise is characterized by a progressive reduction in carbohydrate oxidation and concomitant increase in fat oxidation. Pyruvate dehydrogenase (PDH) controls the entry of pyruvate into oxidative pathways and is a rate-limiting enzyme for carbohydrate metabolism. PDH is controlled by the activities of a kinase (PDK, inhibitory) and phosphatase (stimulatory). To test the hypothesis that increased PDK activity was associated with decreased PDH activity and carbohydrate oxidation during an acute exercise bout, seven recreationally active men completed 4 h of cycle exercise at 55% peak oxygen consumption. Muscle samples were obtained before and at 10 min and 4 h of exercise for the measurement of PDH activity and the extraction of intact mitochondria for the measurements of PDK activity and PDK-2 and PDK-4 protein expression. Carbohydrate oxidation was reduced (P < 0.05) with exercise duration. Muscle glycogen content was lower (P < or = 0.05) at 4 h compared with rest and there was no change in muscle pyruvate content from 10 to 240 min during exercise (10 min: 0.28 +/- 0.05; 240 min: 0.35 +/- 0.09 mmol/kg dry muscle). PDH activity increased (P < 0.05) above resting values at 10 min (2.86 +/- 0.26 mmol.min(-1).kg wet muscle(-1)), but was lower than 10 min after 4 h (2.23 +/- 0.24 mmol.min(-1).kg wet muscle(-1)) of exercise. PDK-2 and PDK-4 protein expression was not different from rest at 10 min and 4 h of exercise. PDK activity at rest averaged 0.081 +/- 0.016 min(-1), was similar at 10 min, and increased (P < 0.05) to 0.189 +/- 0.013 min(-1) at 4 h. Although reduced glycolytic flux may have played a role in decreasing carbohydrate oxidation, the results suggest that increased PDK activity contributed to the reduction in PDH activity and carbohydrate oxidation late in prolonged exercise. The increased PDK activity was independent of changes in intra-mitochondrial effectors, and PDK-2 and PDK-4 protein content, suggesting that it was caused by a change in the specific activity of the existing kinases.     

The control by RNA of pyruvate kinase activity in human skeletal muscle

Summary About 25% of total pyruvate kinase activity in human skeletal muscle is associated with the ribonucleoprotein complexes soluble in salt solutions of high ionic strength. These complexes, called form M_B, crystallize readily from 48% saturated ammonium sulfate at pH 5.6.Crystalline preparations represent a heterogenous population of ribonucleoprotein complexes displaying a graduated activity and a variable RNA content. Free protein was not detected in the preparations.Fractionation of crystalline complexes in salt solutions of varying ionic strength and pH, followed by gel filtration on Sephadex G-200 led to the separation of two nucleoprotein fractions with very high specific activity. Fractions containing 30% RNA and 85% RNA respectively revealed a specific activity of 660–670 U/mg protein at 25°C.Pyruvate kinase form M_A was extracted from muscle homogenate with distilled water, purified to homogeneity and crystallized. It contained less than 0.2% RNA and had a specific activity of 270 U/mg. Active ribonucleoprotein complexes gave in double immunodiffusion test the precipitation bands with the anti-M_A sera at the same protein concentration of both antigens, M_B and M_A.Pyruvate kinase M_B with high activity is sensitive to treatment with RNase. Digestion with RNase for 10 min at 25°C diminished the initial specific activity to about one third. Similar residual activity was found in crystalline ribonucleo protein complexes with low RNA content (3.5–20% RNA) which are resistant to further inactivation by RNase.These results implicate the enhancement and control of pyruvate kinase activity by RNA bound to the enzyme.This work was supported by a grant from the Biochemical and Biophysical Committee of Polish Academy of Sciences.     

Phosphoenolpyruvate hydrolase activity of rabbit muscle pyruvate kinase.

Rabbit muscle pyruvate kinase catalyzes the hydrolysis of P-enolpyruvate at the same active site which catalyzes the physiologically important kinase reaction. The hydrolase activity is lower than the kinase activity by a factor of at least 10(3). There are specific monovalent cation and divalent cation requirements. No other cofactors are required. The relative activation of the pyruvate kinase for the hydrolase reaction is: Ni(II) greater than Co(II) greater than Mg(II) greater than Mn(II). This parallels the rates of nonenzymatic hydrolysis of P-enolpyruvate (Benkovic, S.J., and Schray, K.J. (1968) Biochemistry 7, 4097-4102). The pH rate profiles of the hydrolase and kinase reactions activated by Ni(II) and Co(II) are similar, suggesting common features in their mechanisms. In contrast to the kinase reaction, the reaction velocity of the hydrolase increases at high Co(II) concentrations indicating a second mode for hydrolysis.     

人细胞周期相关激酶启动子的克隆和初步分析

克隆人的细胞周期相关激酶(CCRK)基因启动子,分析与其表达有关的转录调控因子。通过巢式PCR方法,从人的基因组总DNA中分离出CCRK基因5′端非翻译区大小为1072bp的片段。这段片段的3′端起始点在第一个外显子里第一个翻译密码子ATG( 1)上游128bp处。以1072bp为模板,对启动子进行5′端删除分析,分别扩增951bp(-1072／-128)、564bp(-692／-128)、313bp(-441／-128)、127bp(-239／-128)的片段,与pGL3-Basic荧光素酶报告载体重组构建,人工加上克隆位点,5′端带有KpnI、3′端带有XhoI位点。瞬时转染恶性神经胶质瘤细胞株U373,通过双荧光素酶活性进行分析。对分离出的1072bp的片段进行测序,结果与GenBank(Accession AF035013)的序列比较,同源序列达到98％。双荧光素酶活性分析564bp片段有最强的活性,313bp片段为有活性的最小片段。本研究为进一步研究分析CCRK的核心启动子和与其相关的转录因子奠定基础。