The amino acid sequences of a wide range of enzymes that utilize thiamin pyrophosphate (TPP) as cofactor have been compared. A common sequence motif approximately 30 residues in length was detected, beginning with the highly conserved sequence -GDG-and concluding with the highly conserved sequence -NN-. Secondary structure predictions suggest that the motif may adopt a flail fold. The same motif was recognised in the primary structure of a protein deduced from the DNA sequence of a hitherto unassigned open reading frame of Rhodobacter capsulata. This putative protein exhibits additional homology with some but not all of the TPP-binding enzymes.

The reaction catalyzed by adenosine kinase purified from human erythrocytes proceeds via a classical ordered sequential mechanism in which adenosine is the first substrate to bind to and AMP is the last product to dissociate from the enzyme. However, the interpretation of the steady-state kinetic data is complicated by the finding that while AMP acts as a classical product inhibitor at concentrations greater than 5 mM, at lower concentrations AMP can act as an apparent activator of the enzyme under certain conditions. This apparent activation by AMP is proposed to be due to AMP allowing the enzyme mechanism to proceed via an alternative reaction pathway that avoids substrate inhibition by adenosine. Quantitative studies of the protection of the enzyme afforded by adenosine against both spontaneous and 5,5'-dithiobis(2-nitrobenzoic acid)-mediated oxidation of thiol groups yielded "protection" constants (equivalent to enzyme-adenosine dissociation constant) of 12.8 microM and 12.6 microM, respectively, values that are more than an order of magnitude greater than the dissociation constant (Kia = 0.53 microM) for the "catalytic" enzyme-adenosine complex. These results suggest that adenosine kinase has at least two adenosine binding sites, one at the catalytic center and another quite distinct site at which binding of adenosine protects the reactive thiol group(s). This "protection" site appears to be separate from the nucleoside triphosphate binding site, and it also appears to be the site that is responsible for the substrate inhibition caused by adenosine.

A 4175-bp EcoRI fragment of DNA that encodes the a and / 3 chains of the pyruvate dehydrogenase (lipoamide) component (El) of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been cloned in Escherichia coli. Its nucleotide sequence was determined. Open reading frames (pdhA, pdhB) corresponding to the Ela subunit (368 amino acids, M , 41 312, without the initiating methionine residue) and E l p subunit (324 amino acids, M , 35 306, without the initiating methionine residue) were identified and confirmed with the aid of amino acid sequences determined directly from the purified polypeptide chains. The E1p gene begins just 3 bp downstream from the Ela stop codon. It is followed, after a longer gap of 73 bp, by the start of another but incomplete open reading frame that, on the basis of its known amino acid sequence, encodes the dihydrolipoyl acetyltransferase (E2) component of the complex. All three genes are preceded by potential ribosome-binding sites and the gene cluster is located immediately downstream from a region of DNA showing numerous possible promoter sequences. The Ela and E l p subunits of the B. stearotherrnophilus pyruvate dehydrogenase complex exhibit substantial sequence similarity with the Elm and EIP subunits of pyruvate and branched-chain 2-0x0-acid dehydrogenase complexes from mammalian mitochondria and Pseudomonas putida. In particular, the El a chain contains the highly conserved sequence motif that has been found in all enzymes utilizing thiamin diphosphate as cofactor.The 2-0x0-acid dehydrogenase multienzyme complexes catalyse the oxidative decarboxylation of pyruvate, 2-0x0-glutarate and branched-chain 2-0x0 acids, releasing C 0 2 and generating the corresponding acyl-CoA and NADH. The three constituent enzymes of the pyruvate dehydrogenase (PDH) complex are pyruvate dehydrogenase (lipoamide) (El), dihydrolipoamide acetyltransferase (E2) and dihydrolipoamide dehydrogenase (E3). In the PDH complexes from Gram-positive bacteria and mitochondria, the E l components are composed of E l a and E l p polypeptide chains and the E2 components form cores consisting of 60 copies of the E2 polypeptide chains arranged with icosahedral symmetry. These cores bind multiple copies of the E l and E3 components tightly but non-covalently (for recent reviews see [I] and [2]).The E l component catalyses the decarboxylation of pyruvate and the reductive transfer of the acetyl group to a lipoic acid moiety which is covalently bound to the E2

A 2641-bp EcoRI fragment of DNA that encodes the C-terminal part of the dihydrolipoyl acetyltransferase (E2) component and the dihydrolipoamide dehydrogenase (E3) component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus has been cloned in Escherichia coli. Its nucleotide sequence was determined. A 705-bp truncated open reading frame was located at the 5' end of the insert which, together with the 588-bp truncated open reading frame at the 3' end of another EcoRI fragment of B. stearothermophilus DNA previously cloned and sequenced [Hawkins, C. F., Borges, A. & Perham, R. N. (1990) Eur. J. Biochem. 191,, was identified as the gene, pdhC, encoding the E2 polypeptide chain. Direct sequence analysis of the purified E2 chain confirmed that the two EcoRI fragments are adjoining in the B. stearothermophilus genome. The E3 gene, pdhD, begins just 4 bp downstream from the stop codon of the pdhC gene. The amino acid sequences deduced from the pdhC and pdhD genes correspond to proteins of 427 amino acids (E2, M , 46265) and 469 amino acids (E3, M , 49193), respectively. Both genes are preceded by potential ribosome-binding sites and the E3 gene is followed by a stemloop structure characteristic of rho-independent transcription terminators. The B. stearothermophilus E2 and E3 chains exhibit substantial sequence similarity with the corresponding subunits of other 2-0x0-acid dehydrogenase multienzyme complexes. The cloning and sequence analysis described here complete the description of the gene cluster (pdhA, B, C and D ) which encodes the B. stearothermophilus pyruvate dehydrogenase multienzyme complex.The oxidative decarboxylation of 2-0x0-acids is accomplished in most organisms by the 2-0x0-acid dehydrogenase multienzyme complexes, producing the corresponding acyl-CoA and NADH. For the pyruvate dehydrogenase (PDH) complex, the constituent enzymes that function successively in the mechanism are pyruvate dehydrogenase (El), dihydrolipoamide acetyltransferase (E2) and dihydrolipoamide dehydrogenase (E3) (for recent reviews see [I, 21).The PDH complex from the thermophilic, Gram-positive bacterium Bacillus stearothermophilus is assembled round a core of the E2 component of 60 copies of the E2 polypeptide chain organized with icosahedral symmetry [3]. Multiple cop- ies of the El (comprising two polypeptide subunits, Elcl and Elp) and E3 components are attached to this core tightly but non-covalently. The M, of the individual components as well as the morphology of the E2 core [3, 41 closely resemble those of the PDH complexes of eukaryotes. Its thermostability makes the B. stearothermophilus PDH complex a good system for studying the structure of a 2-0x0-acid dehydrogenase complex based on icosahedral symmetry, which differs from the PDH and 2-oxoglutarate dehydrogenase complexes of Escherichia coli in that the latter have octahedral E2 cores [5, 61. The sequence of the first 21 1 amino acid residues from the N-terminus of the B. stearothermophilus E2 chain has been determined [7], showing that this ace...

Summary. Mitochondrial DNA (mtDNA), isolated from worker honeybee larvae, was digested by each of seven 6-base restriction enzymes. Only one enzyme (Bgl II) showed a mtDNA difference between the three tested races (Apis mellifera carcia, A. m. ligustica, A.m. caucasica). Both A.m. carnica and A.m.ligustica showed the same pattern, differing from A.m. caucasica. The degree of fragment pattern similarity revealed that there is only a small level of mtDNA variation between the three races tested. This is in line with previous investigations of enzyme polymorphisms.