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An iterative approach to the a priori determination of the substrate specificity of cAMP- and cGMP-dependent protein kinases (PKA and PKG) by the use of peptide libraries on cellulose paper is described. The starting point of the investigation was an octamer library with the general structure Ac-XXX12XXX, where X represents mixtures of all 20 natural amino acids and 1 and 2 represent individual amino acid residues. The library thus contained all possible 2.56 x 10(10) octamers, divided into 400 sublibraries with defined amino acids 1 and 2 each consisting of 6.4 x 10(7) sequences. After phosphorylation with the kinases in the presence of [gamma-32P]ATP, the sublibrarys Ac-XXXRRXXX and Ac-XXXRKXXX were identified as the best substrates for PKA and PKG, respectively. The second-generation libraries had the structures Ac-XXXRR12X and Ac-XXXRK12X for PKA and PKG and resulted in the most active sequence pools Ac-XXXRRASX and Ac-XXXRKKSX. After delineation of every position in the octameric sequence and extension of the investigation to decameric peptides, the best sequences, Ac-KRAERKASIY and Ac-TQKARKKSNA, were obtained for PKA and PKG, respectively. Promising octameric and decameric peptides were assembled 5 or 10 times each and assayed in order to determine the experimental scatter inherent in the approach. The kinetic data of several octameric and decameric sequences were determined in solution and compared to data for known substrates. The recognition motif of PKA was confirmed by this approach, and a novel substrate sequence for PKG was identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Nearly 100 years ago Michaelis and Menten published their now classic paper (Michaelis, L., and Menten, M. L. (1913) Die Kinetik der Invertinwirkung, Biochemische Zeitschrift 49, 333–369), in which they show that the rate of an enzyme-catalyzed reaction is proportional to the concentration of enzyme-substrate complex predicted by the Michaelis-Menten equation. Because the original text was written in German, yet is often quoted by English speaking authors, we undertook a complete translation of the 1913 publication, which we provide as an online supplement (http://pubs.acs.org). Here we introduce the translation, describe the historical context of the work, and show a new analysis of the original data. In doing so, we uncovered several surprises that reveal an interesting glimpse into the early history of enzymology. In particular, our re-analysis of Michaelis and Menten’s data using modern computational methods revealed an unanticipated rigor and precision in the original publication and uncovered a sophisticated, comprehensive analysis that has been overlooked in the century since their work was published. Michaelis and Menten not only analyzed initial velocity measurements, but they also fit their full time course data to the integrated form of the rate equations, including product inhibition, and derived a single global constant to represent all of their data. That constant was not the Michaelis constant, but rather, Vmax/Km, the specificity constant times the enzyme concentration (kcat/Km*E0).

The formation of a DNA "paper-clip" type triple helix (triplex) with a common sequence 5'-d-(TC)(3)T(a)()(CT)(3)C(b)()(AG)(3) (a and b = 0-4) was studied by UV thermal melting experiments and CD spectra. These DNA oligomers form triplexes and duplexes under slightly acidic and neutral conditions, respectively. The stability of the formed triplexes (at pH 4.5) or duplexes (at pH 7.0 or 8.0) does not vary significantly with the size of the loops (a and b = 1-4). At pH 6.0, the triplex stability is, however, a function of a and b. It is also interesting to note that the oligomer 5'-d-(TC)(3)(CT)(3)(AG)(3) (a and b = 0) forms a stable triplex at pH 4.5 with a slightly lower T(m) value, due to dissociation of a base triad at one end and a distorted base triad at the other, observed by (1)H NMR. Thus, we have here a model system, 5'-d-(TC)(3)T(a)(CT)(3)C(b)(AG)(3), that could form a triplex effectively with (a and b = 1-4) and without (a and b = 0) loops under acidic conditions. In addition, the triplex formation of oligomers with replacement of one, two, or three 2'-deoxycytidine in the Hoogsteen strand by either 2'-deoxypseudoisocytidine (D) or 2'-O-methylpseudoisocytidine (M) was also studied in the sequence 5'-d-(TX)(3)T(2)(CT)(3)C(2)(AG)(3) (where X is C, D, or M). Both CD spectra and UV melting results showed that only D3 [(TX)(3) = (TD)(3)] and M3 [(TX)(3) = (TM)(3)] were able to form the paper-clip structure under both neutral and acidic conditions. This is because the N(3)H of a pseudoisocytosine base can serve as a proton donor without protonation. We hereby proved that the 2'-deoxypseudoisocytidine, similar to 2'-O-methylpseudoisocytidine, could replace 2'-deoxycytidine in the Hoogsteen strand to provide triplex formation at neutral pH.

Lignocellulosic biomass is a potential source of renewable, low-carbon-footprint liquid fuels. Biomass recalcitrance and enzyme cost are key challenges associated with the large-scale production of cellulosic fuel. Kinetic modeling of enzymatic cellulose digestion has been complicated by the heterogeneous nature of the substrate and by the fact that a true steady state cannot be attained. We present a two-parameter kinetic model based on the Michaelis-Menten scheme ( Michaelis, L., and Menten, M. L. ( 1913 ) Biochem. Z. , 49 , 333 - 369 ) with a time-dependent activity coefficient analogous to fractal-like kinetics formulated by Kopelman ( Kopelman, R. ( 1988 ) Science 241 , 1620 - 1626 ). We provide a mathematical derivation and experimental support to show that one of the parameters is a total activity coefficient and the other is an intrinsic constant that reflects the ability of the cellulases to overcome substrate recalcitrance. The model is applicable to individual cellulases and their mixtures at low-to-medium enzyme loads. Using biomass degrading enzymes from cellulolytic bacterium Thermobifida fusca , we show that the model can be used for mechanistic studies of enzymatic cellulose digestion. We also demonstrate that it applies to the crude supernatant of the widely studied cellulolytic fungus Trichoderma reesei ; thus it can be used to compare cellulases from different organisms. The two parameters may serve a similar role to Vmax, KM, and kcat in classical kinetics. A similar approach may be applicable to other enzymes with heterogeneous substrates and where a steady state is not achievable.

The leukocyte cell surface glycoprotein CD45 is a protein tyrosine phosphatase and is involved in signal transduction mediated by the T cell antigen receptor. The asparagine-linked sugar chains were released as oligosaccharides from purified CD45 by hydrazinolysis. Approximately 6 mol of sugar chains was released from 1 mol of CD45. These sugar chains were converted to radioactive oligosaccharides by reduction with NaB3H4 and separated into neutral and acidic fractions by paper electrophoresis. All of the acidic oligosaccharides were converted to neutral ones by digestion with sialidase, indicating that they are sialyl derivatives. Binding of the sialylated oligosaccharides to an SNA-agarose column as well as methylation analysis revealed that the oligosaccharides have only alpha-2,6-linked sialic acid residues. The neutral and sialidase-treated acidic oligosaccharides were fractionated by serial lectin column chromatography followed by Bio-Gel P-4 column chromatography. Structural studies of each oligosaccharide by sequential exo- and endoglycosidase digestion and by methylation analysis revealed that CD45 contains mainly bi-, tri-, and tetraantennary complex-type sugar chains. About 46% of the tetraantennary complex-type sugar chains had the poly(N-acetyllactosamine) groups and 18% of the 2,4-branched triantennary complex-type sugar chains had the fucosyl N-acetyllactosamine group. A portion of the bi- and 2,4-branched triantennary complex-type sugar chains were bisected. In addition to these sugar chains, a small amount of high mannose-type and hybrid-type sugar chains were detected.

Branched polylactosamines on animal cell surfaces are believed to contribute to multivalent interactions in cell adhesion and cell signalling. Their biosynthesis proceeds via linear precursors that become branched by beta1,6-GlcNAc transferases (IGnT6, GlcNAc to Gal). Previous work has identified the tetrasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc (1) and the hexasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1- 3Galbeta1-4GlcNAc (4) as acceptors for a rat serum enzyme activity (cIGnT6), which transfers GlcNAcbeta1-6 units to the midchain galactose residues. Thereby, 1 is converted to the branched pentasaccharide Galbeta1-4GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-4 GlcNAc and 4 to the doubly branched octasaccharide Galbeta1-4GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-+ ++4GlcNAcbeta1-3(GlcNAcb eta1-6)Galbeta1-4GlcNAc [Leppänen, A., Salminen, H., Zhu, Y., Maaheimo, H., Helin, J., Costello, C. E.,Renkonen, O. (1997) Biochemistry 36, 7026-7036]. Here we report that neither the alpha1, 3-fucose-containing derivatives of 1 [Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)G lcNAc and Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Gl cNAc] nor a similar derivative of 4 [Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)+ ++GlcNAcbeta1-3Galbeta1- 4GlcNAc] were acceptors for the rat serum cIGnT6 activity. Hence, the enzyme's branch-forming action was completely prevented at sites in the immediate neighborhood of the fucosylated loci of the polylactosamines. In Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1- 3Galbeta1-4(Fucalpha1-3) GlcNAc, the inhibition of the branch-forming reaction was restricted to the fucose-carrying LacNAc unit; at the middle LacNAc, the branching proceeded normally. However, in the isomeric Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1- 4GlcNAcbeta1-3Galbeta1-4 GlcNAc, the fucose residue prevented branching completely at the middle LacNAc and almost completely at the reducing end LacNAc. In summary, alpha1,3-fucose residues in polylactosamine chains inhibited the cIGnT6 reaction in a site-specific manner, at the fucosylated LacNAc unit itself and also at sites one and two LacNAc units upstream, but not at the LacNAc units downstream from the fucosylated locus. These data imply that site-directed branching in polylactosamines is possible in vitro with the aid of specifically positioned alpha1,3-fucosyl units, that can be removed afterward without harming the branched backbones.

Leu-CAMs (CD11/CD18) consisting of LFA-1, Mac-1, and p150/95 are leukocyte cell surface glycoproteins that are involved in various leukocyte functions. The asparagine-linked sugar chains were released as oligosaccharides from Leu-CAMs by hydrazinolysis. About 12 mol of sugar chains was released from 1 mol of Leu-CAMs. These sugar chains were converted to radioactive oligosaccharides by reduction with sodium borotritide and separated into neutral and acidic fractions by paper electrophoresis. All of the acidic oligosaccharides were converted to neutral ones by digestion with sialidase, indicating that they are sialyl derivatives. The neutral and sialdase-treated acidic oligosaccharides were fractionated by chromatography on lectin columns followed by Bio-Gel P-4 column chromatography. Structural studies of each oligosaccharide by sequential exo- and endoglycosidase digestion and by methylation analysis revealed that Leu-CAMs contain mainly high mannose type and high molecular weight complex type sugar chains. The latter sugar chains were of bi-, tri-, and tetraantennary complex types with the Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----and/or the Gal beta 1----3GlcNAc beta 1----groups together with the Gal beta 1----4GlcNAc group in their outer-chain moieties. In addition to these sugar chains, a small amount of monoantennary complex type and hybrid type sugar chains was found in Leu-CAMs. Furthermore, analysis of the asparagine-linked sugar chains released from the beta-subunit of Leu-CAMs by a series of lectin chromatography showed that subunit-specific glycosylation is not observed between the alpha- and beta-subunits of Leu-CAMs.

When Pseudomonas putida ATCC 39167 and plant-deleterious Pseudomonas fluorescens A225 were grown in an iron-deficient culture medium, they each produced two different novel yellow-green fluorescent pseudobactins: P39167-I, II and PA225-I, II. Pseudobactin P39167-I has a molecular formula of C46H65O23N13 and is monoanionic at neutral pH. P39167-II has the molecular formula of C46H63O22N13 and no charge at neutral pH. Pseudobactin PA225-I has a molecular formula of C46H65O24N13 and is monoanionic at neutral pH whereas pseudobactin PA225-II has the molecular formula of C46H63O23N13 and no charge at neutral pH. All four of the pseudobactins contain a dihydroxyquinoline-based chromophore. The amino acid sequence for the octapeptide in case of pseudobactins from P. putida ATCC 39167 is Chr-Ser(1)-Ala(1)-AcOHOrn-Gly-Ala(2)-OHAsp-Ser(2)-Thr. In case of pseudobactins from P. fluorescens A225, the octapeptide has the sequence Chr-Ser(1)-Ala-AcOHOrn-Gly-Ser(2)-OHAsp-Ser(3)-Thr. For all four pseudobactins (P39167-I, II and PA225-I, II), the serine(1) residue of the octapeptide is attached to the carboxylic acid group on the C-11 of the fluorescent quinoline via an amide bond. Additionally, for pseudobactin P39167-II and PA225-II, the hydroxyl group of the serine(1) residue is also attached to the carboxyl group of threonine residue at the carboxy terminus of the peptide via an ester bond, resulting in a cyclic depsipeptide in contrast to the linear peptide chain of P39167-I and PA225-I. For all four pseudobactins, a malamide group is attached to the C-3 of the quinoline derived chromophore. The three bidentate iron(III) chelating groups in all four pseudobactins consist of a 1,2-dihydroxy aromatic group of the fluorescent chromophore, a hydroxy acid group of beta-hydroxy aspartic acid, and a hydroxamate group from the acylated Ndelta-hydroxyornithine. The amino acid constituents of the pseudobactins P39167 I, II are the same as those in pseudobactin A214, whereas those in A225 I, II are the same as in 7SR1, but in both cases the sequences are different. The uptake results indicate a single outer membrane receptor protein for ferric-pseudobactins in both organisms. The receptor proteins in the two species are similar but not identical.

Neutral oligosaccharides, which accounted for 74% of the total N-linked sugar chains released by hydrazinolysis of rat small intestinal aminopeptidase N, were investigated on a structural basis. They are mainly composed of complex-type sugar chains with tri- and tetraantennary structures, and small amounts of high mannose type sugar chains (7% of the total neutral sugar chains) are also included. The unique feature of the complex-type sugar chains is the most of them contain terminal N-acetylglucosamine residues and blood group H antigenic determinants in their outer chain moieties, and bisecting N-acetylglucosamine residues in their trimannosyl cores. Both the type 1H and type 2H determinants are found, but the former is mainly expressed at the distal portions of the outer chain moieties of the oligosaccharides.

The carbohydrates of BHA, a solubilized hemagglutinin of influenza virus by bromelain digestion, were quantitatively released as oligosaccharides by hydrazinolysis. The oligosaccharide mixture was separated into a neutral and two acidic fractions by paper electrophoresis. Both acidic fractions were resistant to sialidase digestion but were slowly converted to the neutral fraction by incubation with sulfatases. The neutral fraction which comprised about 80% in molar ratio of total oligosaccharides was separated into 13 oligosaccharides by paper chromatography and by Con A-Sepharose column chromatography. Structural studies of these oligosaccharides by sequential exoglycosidase digestion and by methylation analysis revealed that BHA contains a series of high mannose type and bi-, tri-, and tetraantennary complex type sugar chains. Occurrence of Gal beta l leads to 3GlcNAc outer chain in two and bisectional N-acetylglucosamine in one of the biantennary sugar chains is an interesting characteristic of the sugar chains of BHA.

The respiratory NADH dehydrogenase of Escherichia coli has been synthesized in vitro in a coupled transcription--translation system with cloned deoxyribonucleic acid (DNA) as template. The identity of the protein produced was confirmed by paper chromatography and electrophoresis of tryptic peptides. [35S]Methionine-labeled tryptic peptides from the in vitro product were shown to comigrate with authentic methionine-containing tryptic peptides from the purified enzyme. Using a transcription-translation system derived from an ndh mutant, it was shown that the enzyme produced in vitro was incorporated into membrane vesicles of the mutant to give functional, cyanide-sensitive NADH oxidase activity. Radiochemical N-terminal sequencing of the synthesized NADH dehydrogenase showed that the product was a mixture of three different species, with N-formylmethionine, methionine, or threonine at the N terminus. The results indicated that only partial N-terminal processing was occurring in vitro and that the first residue of the unprocessed NADH dehydrogenase is N-formylmethionine. Since DNA sequencing has shown that this residue is encoded by UUG [Young, I. G., Rogers, B. L., Campbell, H. D., Jaworowski, A., & Shaw, D. C. (1981) Eur. J. Biochem. (in press)], this work verifies the role of UUG as a normal initiation codon.

The chemical synthesis of lactose operator DNA segments is described. The 31-base-paired duplex contains the DNA recognized by lac repressor protein and twofold rotationally symmetric base pairs on either side of the tight binding region. The synthesis includes the deoxyoligonucleotides d(T-G-T-G-G), d(A-A-T-T-G-T-G-A-G), d(C-G-G-A-T-A-A-C-A-A-T-T), d(T-C-A-C-A), d(T-G-T-G-A-A-A-T-T-G-T), d(T-A-T-C-C-G-C-T-C-A-C), and d(A-A-T-T-C-C-A-C-A). These deoxyoligonucleotides were characterized by two-dimensional sequencing techniques, paper chromatography, and thin-layer chromatography.

Purified beta subunit of human chorionic gonadotropin (hCG) was partially reduced with beta-mercaptoethanol, carboxymethylated, and digested with chymotrypsin. The peptides were isolated by high-voltage electrophoresis and paper chromatography. Five major disulfide-containing peptides were isolated, and their location in the parent molecule was established by amino acid composition and amino- and carboxy-terminal analyses. All of these peptides inhibited the binding of 125I-labeled hCG by anti-beta hCG serum. The inhibitory effect of these peptides was lost when their disulfide bonds were reduced and alkylated. Synthetic carboxy-terminal peptides were not inhibitory. Based on these data it is concluded that a major antigenic site of hCG resides in the region of residues 21-23 with a disulfide bond connecting cysteine-23 or -26 with the cysteines at positions 72 or 110.

Uridine 5'-(5-thio-alpha-D-glucopyranosyl pyrophosphate), UDPTG, appears to be a potent activator of rat liver glycogen synthetase a even though it is not a substrate. At 1.0 mM, UDPTG causes over 400% activation of glycogen synthetase a activity. Activation by UDPTG is accompanied by normalization of the otherwise sigmoidal kinetics for UDPG with glycogen synthetase a and a decrease in the apparent Km for UDPG from approximately 2.0 to 0.62 mM. UDPTG inhibits catalytic activity at higher concentrations. At the concentrations examined, UDPTG has no effect on glycogen synthetase b activity. The use of glycogen synthetase free from glycogen synthetase b phosphatase and the selective inhibition of glycogen synthetase b phosphatase by 100 mM NaF-indicate that conversion of synthetase b to a is not responsible for the activation. Moreover, the use of the colorimetric assay for glycogen synthetase activity precludes effects of UDPTG on glycogen phosphorylase activity. UDPTG, chemically synthesized in 60% yield, is characterized by chromatographic and electrophoretic procedures, by its uv spectra, and by analysis of products after chemical and enzymatic hydrolysis.

Asparagine-linked sugar chains of rat kidney aminopeptidase N and dipeptidylpeptidase IV were investigated comparatively. Oligosaccharides released from the two enzymes by hydrazinolysis were fractionated by paper electrophoresis, serial chromatographies on columns of immobilized Aleuria aurantia lectin, concanavalin A, phytohemagglutinin E4, and Datura stramonium agglutinin, and Bio-Gel P-4 (less than 400 mesh) column chromatography. Structures of oligosaccharides in each fraction were assumed by their effective molecular sizes and behaviors on the four lectin columns and then confirmed by sequential exoglycosidase digestion and methylation analysis. The sugar chains of aminopeptidase N and dipeptidylpeptidase IV are almost the same as those of rat kidney gamma-glutamyltranspeptidase reported previously [Yamashita, K., Hitoi, A., Matsuda, Y., Tsuji, A., Katunuma, N., & Kobata, A. (1983) J. Biol. Chem. 258, 1098-1107]. They are a mixture of 5 high mannose type sugar chains and 32 (for aminopeptidase N) or 26 (for dipeptidylpeptidase IV) mono-, bi-, tri-, and tetraantennary complex type sugar chains. The unique feature of the complex-type sugar chains of both enzymes is that they all contain the bisecting N-acetylglucosamine residue and are incompletely galactosylated in their outer-chain moieties.

The naturally occurring modified nucleoside, N-[(9-beta-D-ribofuranosylpurin-6-yl)-N-methylcarbamoyl]-L-threonine (mt6A), and the corresponding glycine analog mg6A were synthesized from N6-methyl-2',3',5'-tri-O-acetyladenosine and the appropriately blocked isocyanates derived from threonine and glycine. The natural mt6A isolated from Escherichia coli tRNA (F. Kimura-Harada et al. (1972), Biochemistry 11, 3910), from wheat embryo tRNA (R. Cunningham and M. W. Gray (1974), Biochemistry 13, 543), and from rat liver tRNA (Rogg et al. (1975), Eur. J. Biochem. 53, 115) was found to be identical with the synthetic mt6A in paper and thin-layer chromatography and electrophoresis. Several analogs of the parent 6-ureidopurine ribonucleoside, N-[(9-beta-D-ribofuranosylpurin-6-yl)carbamoyl]-L-thronine (t6A), were also prepared. Starting from 2',3',5'-tri-O-acetylguanosine and 2',3',5'-tri-O-acetylcytidine and the above isocyanates, the t6A analogs, N-[(9-beta-D-ribofuranosyl-6-oxo-1H-purin-2-yl)carbamoyl]-L-threonine (t2G) and N-[(1-beta-D-ribofuranosyl-2-oxypyrimidin-4-yl)carbamoyl]-L-threonine (t4C), were prepared. Also synthesized were the corresponding glycine analogs, g2G and g4C, from guanosine and cytidine, respectively. The 2'-deoxyribosyl analog, N-[(9-beta-D-2'-deoxyribofuranosylpurin-6-yl)carbamoyl]-L-threonine (2'-deoxy-t6A), and the arabinosyl derivative, N-[(9-beta-D-arabinofuranosylpurin-6-yl)carbamoyl]-L-threonine (t6AraA), were synthesized from the appropriate urethane and the requisite amino acid. The ureido group in mt6A could not be hydrolyzed by the enzymes urease, peptidase, and protease. Various chemical and biological properties of the naturally occurring mt6A and the related analogs are discussed.

Enzyme I, the phosphoenolpyruvate:protein phosphotransferase (EC 2.7.3.9), which is part of the bacterial phosphoenolpyruvate- (PEP) dependent phosphotransferase system, has been purified from Streptococcus faecalis by using a large-scale preparation. Size exclusion chromatography revealed a molecular weight of 140 000. On sodium dodecyl sulfate gels, enzyme I gave one band with a molecular weight of 70 000, indicating that enzyme I consists of two identical subunits. The first 59 amino acids of the amino-terminal part of the protein have been sequenced. It showed some similarities with enzyme I of Salmonella typhimurium. The active center of enzyme I has also been determined. After phosphorylation with [32P]PEP, the enzyme was cleaved by using different proteases. Labeled peptides were isolated by high-performance liquid chromatography on a reversed-phase column. The amino acid composition or amino acid sequence of the peptides has been determined. The largest labeled peptide was obtained with Lys-C protease and had the following sequence: -Ala-Phe-Val-Thr-Asp-Ile-Gly- Gly-Arg-Thr-Ser-His*-Ser-Ala-Ile-Met-Ala-Arg-Ser-Leu-Glu-Ile-Pro-Ala- Ile-Val-Gly-Thr-Lys-. It has previously been shown that the phosphoryl group is bound to the N-3 position of a histidyl residue in phosphorylated enzyme I. The single His in position 12 of the above peptide must therefore carry the phosphoryl group.