Amino Acid Composition Of Enzymes Research Articles

Relationships between metabolic fluxes and enzyme amino acid composition

AbstractMetabolic fluxes are a key parameter of metabolic pathways being closely related to the kinetic properties of enzymes and could be conditional on their sequence characteristics. This study examines possible relationships between the metabolic fluxes and the amino acid (AA) composition (AAC) for enzymes from the yeast Saccharomyces cerevisiae glycolysis pathway. Metabolic fluxes were quantified by the COPASI tool using the kinetic models of Hynne and Teusink at 25 mM, 50 mM, and 100 mM of external glucose or employing literature data for cognate kinetic or stoichiometric models. The enzyme sequences were taken from the UniProtKB, and the AAC computed by the ExPASy/ProtParam tool. Multiple linear regressions (89.07% < R2 adjusted < 91.82%; P<0.00001) were found between the values of metabolic fluxes and the selected sets of AA frequencies (5 to 7 for each model). Selected AA differed from the rest by their physicochemical and structural propensities, thus suggesting a distinctive contribution to the properties of enzymes, and hence the metabolic fluxes. The results provide evidence that metabolic fluxes of the yeast glycolysis pathway are closely related to the AAC of relevant enzymes and support the view that catalytic, binding and structural residues are interdependent to ensure the efficiency of biocatalysts.

Relationships between kinetic constants and the amino acid composition of enzymes from the yeast Saccharomyces cerevisiae glycolysis pathway

The kinetic models of metabolic pathways represent a system of biochemical reactions in terms of metabolic fluxes and enzyme kinetics. Therefore, the apparent differences of metabolic fluxes might reflect distinctive kinetic characteristics, as well as sequence-dependent properties of the employed enzymes. This study aims to examine possible linkages between kinetic constants and the amino acid (AA) composition (AAC) for enzymes from the yeast Saccharomyces cerevisiae glycolytic pathway. The values of Michaelis-Menten constant (KM), turnover number (kcat), and specificity constant (ksp = kcat/KM) were taken from BRENDA (15, 17, and 16 values, respectively) and protein sequences of nine enzymes (HXK, GADH, PGK, PGM, ENO, PK, PDC, TIM, and PYC) from UniProtKB. The AAC and sequence properties were computed by ExPASy/ProtParam tool and data processed by conventional methods of multivariate statistics. Multiple linear regressions were found between the log-values of kcat (3 models, 85.74% < Radj.2 <94.11%, p < 0.00001), KM (1 model, Radj.2 = 96.70%, p < 0.00001), ksp (3 models, 96.15% < Radj.2 < 96.50%, p < 0.00001), and the sets of AA frequencies (four to six for each model) selected from enzyme sequences while assessing the potential multicollinearity between variables. It was also found that the selection of independent variables in multiple regression models may reflect certain advantages for definite AA physicochemical and structural propensities, which could affect the properties of sequences. The results support the view on the actual interdependence of catalytic, binding, and structural residues to ensure the efficiency of biocatalysts, since the kinetic constants of the yeast enzymes appear as closely related to the overall AAC of sequences.

Enzymes in atlatoxin B1 biosynthesis: Strategies for identifying pertinent genes

Recent work on the aflatoxin biosynthetic pathway is reviewed, with special emphasis on the enzymes of the late stages of the pathway involving conversion of sterigmatocystin (ST) to aflatoxin B1 (AFB1) through an O-methylsterigmatocystin intermediate. Two enzyme activities were discovered in subcellular fractions of cell-free extracts of a mutant strain of Aspergillus parasiticus (SRRC 163): 1) A post-microsomal methyltransferase (MT) catalyzed conversion of ST to OMST, and 2) a microsomal-associated activity (oxido-reductase) converted OMST to AFB1. The 168 KDa, anionic MT was purified to homogeneity and characterized (two subunits, 110 KDa and 58 KDa). Preliminary evidence indicated the presence of a cationic isozyme of the MT in mycelial extracts. The oxido-reductase has been partially purified and characterized. Polyclonal antibodies were prepared to the anionic MT and the enzyme's amino acid composition determined. A cDNA library has been constructed from mRNA isolated from Aspergillus parasiticus mycelia during the onset of AFB1 biosynthesis for the purpose of identifying the genes responsible for aflatoxin biosynthesis.

Comparative studies on AMP-deaminase—IV. Amino acids composition of the enzymes from rat, rabbit, hen, frog, pikeperch skeletal muscle and frog liver

1. 1. Amino acids analysis showed a great degree of homology between vertebrate muscle AMP-deaminases. 2. 2. Significant differences were found in the amino acid composition of frog liver AMP-deaminase and skeletal muscle enzymes. 3. 3. A significant amount of glucosamine was found only in the rabbit red muscle enzyme. 4. 4. Frog liver AMP-deaminase had an isoelectric point distinct from the isoelectric points of muscle enzymes.

Purification and properties of the extracellular Metallo-proteinases of Chromobacterium lividum (NCIB 10926)

Four extracellular proteolytic enzymes (I–IV) (EC 3.4.22.—) were identified in static cultures of Chromobacterium lividum (NCIB 10926) by agar gel electrophoresis and isoelectric focusing. Proteinases I–III were freed of nonenzymic protein by chromatography on TEAE-cellulose and CM-cellulose. The enzyme mixture was then fractionated in a pH gradient by isoelectric focusing. All three enzymes were shown to be heat-labile metallo-enzymes. Optimal activity occurred at pH 5.6 for enzyme I and at pH 6.2 for enzymes II and III. Remazolbrilliant Blue-hide powder was a sensitive substrate for these enzymes. Proteinase I was also shown to degrade haemoglobin and casein effectively, but not myoglobin, ovalbumin or bovine serum albumin. Proteinases I–III exhibited molecular weight values of 75 000, 72 000 and 67 000 by exclusion chromatography and 71 000 and 66 000 by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis for enzyme I and II, respectively. The amino acid compositions of enzymes I and II were somewhat similar. Proteinase I was inhibited by EDTA, 1,2-di(2-aminoethoxy)ethane- N, N, N′, N′-tetraacetic acid or 1,10-phenanthroline and both Ca 2+ and Co 2+ were required for maximal activity. Mg 2+ could substitute for Ca 2+ or Mn 2+ for Co 2+. The interrelationship of proteinases I–III is discussed.