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A protein is usually classified into one of the following four structural classes: all α, all β, (a + B) and α/β. In this paper, based on the maximum correlation-coefficient principle, a new formulation is proposed for predicting the structural class of a protein according to its amino acid composition. Calculations have been made for a development set of proteins from which the amino acid compositions for the standard structural classes were derived, and an independent set of proteins which are outside the development set. The former can test the self consistency of a method and the latter can test its extrapolating effectiveness. In both cases, the results showed that the new method gave a considerably higher rate of correct prediction than any of the previous methods, implying that a significant improvement has been achieved by implementing the maximum-correlation-coefficient principle in the new method. [ABSTRACT FROM AUTHOR]

Monoamine oxidases (MAO) A and B show a high degree of amino acid similarity. Apart from the NH2-terminus, which contains an ADP-binding consensus sequence, little is known about their structural features or the sequences involved in the binding of substrates. In the present paper, we have studied the structure/function relationships of MAOs by constructing 18 different chimeric forms of MAO, engineered by moving progressively the junction between the NH2-terminus of one MAO form with the COOH-terminus of its isoenzyme. After transient expression in HEK-293 cells, the properties of these chimeric enzymes were investigated using both selective and nonselective substrates and inhibitors. Whereas exchange of the ADP-binding sequence did not modify the catalytic properties of either MAO isoforms, chimeras with increasing length of the NH2-terminus of MAO-A (up to residue 256) showed a marked decrease in affinity towards the MAO-B substrate phenylethylamine and the inhibitor N-(2-aminoethyl)-5-chloro-2-pyridine carboxamide · HCI (lazabemide) when compared to wild-type MAO-B. No major changes were observed in the kcat, values of these chimeras. From the data obtained, two sequences, i.e. 62–103 and 146–220, appeared of particular importance in constituting the binding site of MAO-B. On the other hand, the catalytic properties and specificity of MAO-A appeared to be relatively insensitive to substitution of both the NH2-- (up to position 112) and COOH-termini (from residue 395) of MAO-A with the corresponding MAO-B sequences. However, further modification of the central 283-residue sequence of MAO-A did not appear compatible with enzymic activity. None of the engineered chimeras showed a shift in specificity from one isoform to the other. [ABSTRACT FROM AUTHOR]

1. A basic protein (pI ≈ 9.0) exhibiting superoxide dismutase activity was purified to homogeneity from rat liver by DEAE-cellulose, CM-cellulose and S-hexylglutathione affinity gel chromatography, chromatofocusing and Sephadex G-150 gel filtration. 2. The purified enzyme had specific activity of 4700 units/mg protein. The activity was not affected by 2 mM KCN. Manganese was detected in the enzyme preparation; the content was 0.9 mol/mol subunit. The N-terminal sequence of the first 23 amino acids of the enzyme exhibited a strong homology (except at position 11) with the mature protein of human Mn-superoxide dimutase. It is, therefore, concluded that the purified enzyme is Mn- superoxide dismutase. 3. The N-terminal amino acid sequence showed that about 50% of tyrosine at position 11 was substituted by glutamine, suggesting the existence of microheterogeneity of the superoxide dismutase protein. 4. The superoxide dismutase purified here was found to consist of subunits with an apparent relative molecular mass of 25000. This is larger than the value hitherto reported for rat liver Mn-superoxide dismutase (Mr, 22400); the previous low value is attributed to differences in methods. 5. The enzyme was shown by immunoblotting to be exclusively Localized in the mitochondrial fraction in the liver. The tissue content of Mn-superoxide dismutase is organaspecific, and was the highest in heart. The precursor protein of the Mn-superoxide dismutase was not detectable in the liver cytosolic and mitochondrial fractions as well as in several extrahepatic organs (lung, heart, brain, muscle, kidney and testis), suggesting rapid transport across mitochondrial membranes and processing of the superoxide dismutase protein. [ABSTRACT FROM AUTHOR]

Information on the regulation and structure–function relation of enzymes involved in DNA precursor synthesis is pivotal, as defects in several of these enzymes have been found to cause depletion or deletion of mitochondrial DNA resulting in severe diseases. Here, the effect of amino acid 106 on the enzymatic properties of the cell-cycle-regulated human cytosolic thymidine kinase 1 (TK1) is investigated. On the basis of the previously observed profound differences between recombinant TK1 with Val106 (V106WT) and Met106 (V106M) in catalytic activity and oligomerization pattern, we designed and characterized nine mutants of amino acid 106 differing in size, conformation and polarity. According to their oligomerization pattern and thymidine kinetics, the TK1 mutants can be divided into two groups. Group I (V106A, V106I and V106T) behaves like V106WT, in that pre-assay exposure to ATP induces reversible transition from a dimer with low catalytic activity to a tetramer with high catalytic activity. Group II (V106G, V106H, V106K, V106L and V106Q) behaves like V106M in that they are permanently high activity tetramers, irrespective of ATP exposure. We conclude that size and conformation of amino acid 106 are more important than polarity for the catalytic activity and oligomerization of TK1. The role of amino acid 106 and the sequence surrounding it for dimer–tetramer transition was confirmed by cloning the putative interface fragment of human TK1 and investigating its oligomerization pattern. [ABSTRACT FROM AUTHOR]

Nucleoside-diphosphate (NDP) kinase was purified from crude extracts of Streptomyces coelicolor to over 90% homogeneity in a single step using an adenosine 3',5'-cyclic monophosphate (cAMP) binding column. The specific activity of protein in the fraction eluted with cAMP (400 U/mg) was about 3600- Md higher than that in the crude extract. This enzyme was autophosphorylated in the presence of [γ- 12P]ATP. The high-energy phosphoenzyme intermediate was stable in alkali and highly labile in acid; this suggests the presence of an N-phosphate amino acid (most probably a histidine residue). A tetrameric form of the 15-kDa protein was suggested by its apparent molecular mass (66 kDa) on a gel filtration column. The measured Michaelis constant (Km) for ATP was 85 μM. The IC50, for cAMP of 6 mM suggested weak competitive inhibition. However. no evidence that cAMP acts as an allosteric effector was obtained. The ndk gene from S. coelicolor was isolated and sequenced. The deduced amino acid sequence was very similar to other NUP kinases. However, unique characteristics were also noted, including a truncated C-terminus that makes it one of the smallest NDP kinases reported in the literature. [ABSTRACT FROM AUTHOR]

A new lipase from Penicillium carnembertii U-150, which is specific for monoacylglycerols and diacylglycerols, but not triacylglycerols, was purified as four active components using concanavalinA-Sepharose column chromatography, crystallized in the form of needles, and its properties investigated. No significant difference was observed in substrate specificity, but molecular mass and other enzymatic properties, such as pH, heat stability and optimum pH and temperature, were clearly different between the unadsorbed and the three adsorbed components on concanavalinA-Sepharose; the three adsorbed components were similar to each other and more stable than the unadsorbed component. On the other hand, after enzymatic removal of carbohydrates from the three adsorbed components, their enzymatic properties became similar to those of the unadsorbed component. The carbohydrates of this lipase contribute to the stability of the enzyme, but not to its enzyme activity. The amino acid compositions of the four components did not differ from each other, and tryptic mapping of the deglycosylated components and amino acid composition of the tryptic fragments were identical. The carbohydrate compositions of four intact components were, however, different from each other. Ail four components have the same polypeptide backbone and multiple forms of this lipase are due to the differences in composition of the carbohydrates bound in this lipase. [ABSTRACT FROM AUTHOR]

The amino acid sequence of glycogenin from rabbit skeletal muscle has been determined. The N-acetylated protein consists of 332 amino acids and has a molecular mass of 37278 Da. The novel tyrosyl-glucose linkage between glycogenin and glycogen [Smythe, C., Caudwell, F. B., Ferguson, M. & Cohen, P. (1988) EMBO J. 7, 2681 -2686] is shown to occur at a single site, tyrosine-194. Although glycogenin is a UDPaGIc utilising glucosyltransferase that self-glucosylates [Pitcher, J., Smythe, C. & Cohen, P. (1988) Eur. J. Biochem. 176, 391 - 395], following addition by an unknown enzyme of the first glucose to tyrosine-194, it is not homologous to either human glycogen synthase or other UDP-Glc-requiring enzymes. [ABSTRACT FROM AUTHOR]

An NAD(P)H-dependent reductase able to reduce a new class of cyclic unsaturated compounds named ketimines has been detected and purified 2500-fold from pig kidney. Some molecular and kinetic properties of this enzyme have been determined. The enzymatic reduction proceeds with a classical ping-pong mechanism and some results suggest that the true substrate has the ketiminic structure and is in equilibrium with the enaminic and keto-open forms. As previously described, ketimines arise from the deamination of a number of sulfur-containing amino acids, i. e. L-Cystathionine, L-lanthionine and s-aminoethyl-L-cysteine, catalyzed by a widespread mammalian transaminase. The enzymatic reduction products of ketimines have been identified as cyclothionine, 1,4-thiomorpholine 3,5-dicarboxylic acid and 1,4-thiomorpholine 3-carboxylic acid. Some of these compounds have been detected in mammals, thus suggesting a possible rote of this enzyme in their biosynthesis. [ABSTRACT FROM AUTHOR]

Two of the four electrophoretic histone H2B variants present in wheat embryos have been isolated. The complete primary structure of the H2B(2) variant has been deduced from sets of overlapping peptides generated by CNBr cleavage, Staphyloccocus aureus V8 protease, endoproteinase Arg-C, the post-proline cleaving enzyme, chymotrypsin and cleavage in dilute acid. A minimum of 17 peptides were required to establish the sequence. This variant has a blocked N terminus and comprises a total of 149 amino acids. The C-terminal two-thirds of the protein are highly homologous to vertebrate H2B. In contrast, the N-terminal third is entirely different and contains an N-terminal extension of 23 residues in which the sequence Ala-Glu-Lys or variants are repeated several times. This region is also highly homologous to the H2B from Tetrahymena pyriformis. It shows in addition similarities to wheat H2A(1) and bovine H1. [ABSTRACT FROM AUTHOR]

The amino acid analog L-cyclohexylalanine, which may be considered an analog of the hydrophobic amino acids leucine and valine, was found to thioester-bind to the heavy enzyme of gramicidin S synthetase. The results indicate that it preferably binds to the thiol site of leucine, although binding to the valine site also occurs. In the synthesis of the cyclic decapeptide by gramicidin S synthetase, the results suggest that L-cyclohexylalanine can replace L-leucine and L-valine. We have also found that in the synthesis of the cyclic decapeptidase L-leucine can replace L-valine and vice versa. The results further indicate that the previously reported thioester binding of D and L-phenylalanine to the heavy enzyme takes place at the thiol site of leucine. [ABSTRACT FROM AUTHOR]

The amino acid composition of octopine dehydrogenase from muscles of Pecten maximus was determined. The calculated partial specific volume of 0.74 allowed us to evaluate the molecular weight of the enzyme as 38000. It is concluded that this enzyme consists of a single polypeptide chain on the basis of the molecular weight determined from equilibrium ultracentrifugation in 6 M guanidine hydrochloride and from electrophoresis on polyacrylamide gel containing sodium dodecylsulfate as well as on the basis of the number of peptides obtained by tryptic digestion. The presence of one essential -SH group per mole of enzyme and of one binding site for NADH are also consistent with the existence of a single active center. No NH2-terminal amino acid can be detected by reaction with dansyl chloride and with phenylisothiocyanate. Controlled carboxypeptidase action showed that the C-terminal amino acid is lysine. [ABSTRACT FROM AUTHOR]

1. Iodoacetamide was found to inactivate pig heart mitochondrial malate dehydrogenase in a pseudo first order reaction. 2. Two moles of 14C-labelled reagent per mole enzyme are incorporated, into the protein. 3. The reactive residue was identified by amino acid analysis and thin layer chromatography as a histidine alkylated at nitrogen 3. [ABSTRACT FROM AUTHOR]