Purification and properties of guanosine triphosphate cyclohydrolase II from Escherichia coli.

An enzyme that uses GTP as substrate for the formation in stoichiometric quantities of formate, inorganic pyrophosphate, and 2,5-diamino-6-hydroxy-4-(ribosylamino)pyrimidine-5'-phosphate has been purified 2200-fold from extracts of Escherichia coli B. This enzyme is named GTP cyclohydrolase II to distinguish it from a previously studied E. coli enzyme, named GTP cyclohydrolase (and called GTP cyclohydrolase I in this paper), that catalyzes the first of a series of enzymatic reactions leading to the biosynthesis of the pteridine portion of folic acid (Burg, A. W., and Brown, G. M. (1968) J. Biol. Chem. 243, 2349-2358). Some of the properties of GTP cyclohydrolase II are: (a) divalent cations are required for activity (Mg2+ is most effective); (b) its molecular weight, estimated by filtration on Sephadex G-200, is 44,000; (c) the K-m for GTP is 41 mum; (d) its pH optimum is 8.5; and (e) its activity is inhibited by inorganic pyrophosphate, one of the products of the reaction. Compounds not used as substrate are: GDP, GMP, guanosine, dGTP, ATP, ITP, and XTP. Properties a, b, c, and e (above), as well as the nature of the products, distinguish this enzyme from GTP cyclohydrolase I. Since GTP cyclohydrolase II apparently is not concerned with the biosynthesis of folic acid, the possible physiological role of this enzyme in the biosynthesis of riboflavin is considered in the light of the present investigations and the previously published work on riboflavin biosynthesis by other investigators.     

Purification and properties of acyl coenzyme A thioesterase II from Rhodopseudomonas sphaeroides

A high molecular weight acyl coenzyme A (acyl-CoA) thioesterase, designated thioesterase II, has been purified 5300-fold from photoheterotrophically grown cells of Rhodopseudomonas sphaeroides. In contrast to R. sphaeroides acyl-CoA thioesterase I [Boyce, S.G., & Lueking, D.R. (1984) Biochemistry 23, 141-147], thioesterase II has a native molecular mass (Mr) of 120,000, is capable of hydrolyzing saturated and unsaturated acyl-CoA substrates with acyl chain lengths ranging from C4 to C18, and is completely insensitive to the serine esterase inhibitor diisopropyl fluorophosphate. Palmitoyl-CoA and stearoyl-CoA are the preferred (lowest Km) saturated acyl-CoA substrates and vaccenoyl-CoA is the preferred unsaturated substrate. However, comparable Vmax values were obtained with a variety of acyl-CoA substrates. Unlike a similar thioesterase present in cells of Escherichia coli [Bonner, W.M., & Bloch, K. (1972) J. Biol. Chem. 247, 3123-3133], R. sphaeroides thioesterase II displays a high ratio of decanoyl-CoA to palmitoyl-CoA activities and exhibits little ability to hydrolyze 3-hydroxyacyl-CoA substrates. Only 3-hydroxydodecanoyl-CoA supported a measurable rate of enzyme activity. With the purification of thioesterase II, the enzymes responsible for greater than 90% of the acyl-CoA thioesterase activity present in cell-free extracts of R. sphaeroides have now been identified.     

Purification and properties of a periplasmic aminoendopeptidase from Escherichia coli.

A periplasmic aminoendopeptidase from Escherichia coli has been purified to hemogeneity. It is a monomer of molecular weight 45000 and containing one -- SH group that is necessary for catalytic activity. The study of its substrate specificity indicated that the enzyme has both aminopeptidase and endopeptidase activity. The pH optimum for L-alanine p-nitroanilide hydrolysis is between 7 and 7.5 and that for 125I-labeled casein proteolysis between 7.3 and 7.6. The activation energy for the hydrolysis of L-anine p-nitroanilide was calculated to be 5.3 kcal X mol-1 (22.2 kJ X mol-1).     

Purification and some novel properties of Escherichia coli RNase II.

RNase II of Escherichia coli (EC 3.1.4.23) has been purified to apparent homogeneity. The K+-activated diesterase activity against poly(U), which defines RNase II, cochromatographs with activity against T4 mRNA or pulse-labeled E. coli RNA successively on DEAE-cellulose, hydroxyapatite or phosphocellulose, and Sephadex G-150 columns. Activities with both substrates are selectively reduced to less than 2% of the wild type level in a newly isolated mutant strain, S296, or after thermal inactivation in a mutant strain with temperature-sensitive RNase II. RNase II releases 5'-XMP without a lag as its only detectable alcohol-soluble produce from all substrates and has an apparent molecular weight of 80,000 to 90,000 in both nondissociating and sodium dodecyl sulfate-polyacrylamide gels. The pure enzyme shows the standard K+ activation against poly(A), poly(U), or poly(C), but only a slight preference for K+ over Na+ ions with T4 mRNA or pulse labeled E. coli RNA as substrate. Uniformly labeled E. coli rRNA or tRNA is degraded little if at all.