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Transfer of the acetyl- l -leucine (AcLeu) residue from AcLeu-pentanucleotide to synthetic substrates under conditions of the fragment reaction was used to study the specificity of the acceptor site of ribosomal peptidyl transferase. 2′(3′)-O-Aminoacyl nucleosides are the simplest acceptor substrates. Their acceptor activity is dependent on the nature of the nucleoside to which the amino acid residue is bound. The acceptor activity decreased in the sequence 2′(3′)-O- l -phenylalanyladenosine (A-(Phe)) > I-(Phe) > C-(Phe); U-(Phe) was inactive. The presence of a free 2′-hydroxyl group in the ribose moiety of the aminoacyl adenosines was important for the acceptor activity, as shown by the low activity of dA-(Phe) in comparison with A-(Phe). Acceptor activity was influenced by the nature of the side chain of the amino acid residue bound to adenosine: A-(Phe) was a more active acceptor than puromycin, while the acceptor activity of 2′(3′)-O-glycyladenosine A-(Gly) was very low. Free phenylalanine, phenylalanine methyl ester, and adenosine did not act as acceptors. As terminal products of the reactions of AcLeu-pentanucleotide with puromycin, A-(Phe), I-(Phe) and C-(Phe), we isolated AcLeu-puromycin, 2′(3′)-O- acetyl- l -leucyl- l -phenylalanyladenosine (A-(AcLeu-Phe)), I-(AcLeu-Phe) and C-(AcLeu-Phe), respectively.

The binding of vitamin B12 by serum proteins was studied by separating Co57B12-enriched serum by Sephadex gel filtration, column chromatography with DEAE-cellulose, and paper electrophoresis. Each method of separation yielded two discrete B12-binding fractions. However, the analysis of each serum by all three separation technics indicated that one of the fractions was, in each case, bipartite. The "high" molecular weight B12-binding fraction defined by Sephadex gel filtration consisted of transcobalamin I and just part of the transcobalamin II fraction. The remaining portion of transcobalamin II was eluted from Sephadex gel in a "low" molecular weight fraction. Thus, transcobalamin II, equivalent to the β-globulin B12-binder, consisted of both "high" and "low" molecular weight components. This suggests that there are at least three serum proteins that can bind vitamin B12: two β-globulins, together comprising the transcobalamin II fraction and differing in molecular weight; and transcobalamin I.

The active site cysteinyl peptide isolated from a tryptic digest of rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) by column chromatography has been found to contain 15 amino acid residues, of which 7 are hydrophobic. The tryptophyl peptides in such digests also appear to consist predominantly of residues which are hydrophobic.

The active thiol group of lombricine kinase from Lumbricus terrestris muscle was labelled with N-ethyl-[1-14C]maleimide. The resulting inactivated N-ethyl-[1-14C]succinimido enzyme was then subjected to tryptic hydrolysis. The peptide containing the labelled essential thiol group was isolated and found to contain: Leu-Gly-Tyr-Ile-Thr-[14C]Cys-Pro-Gly-Ser-Asn-Leu-Gly-Thr-Leu-Arg. The amino acid sequence around this thiol group was very similar with that of homologous ATP: guanidine phosphotransferases previously studied, arginine kinase from Homarus vulgaris muscle, creatine kinases from ox brain and ox muscle and from rabbit muscle. In addition among the other enzymes of this group, lombricine kinase is of special interest since it is the only dimeric enzyme of molecular weight ≃ 80000 which possesses only one essential thiol group and one nucleotide binding site per two subunits.

The size of the active site of carboxypeptidase-A was investigated by studying the kinetics of hydrolysis of peptides of L-alanine, D-alanine and L-phenylalanine, as well as of a number of their N-benzyloxycarbonyl, N-acetyl, N-phenylproprionyl and N-methyloxycarbonyl derivatives. From a comparison of the various kinetic parameters ( K m, kcat) it was concluded that the active site of this enzyme extends over about 18 A. The binding area can be divided into 5 “subsites”, each accommodating one amino acid residue (or blocking group) of the substrate. By comparing K m values of pairs of substrates containing either a methyl or a benzyl side-chain in equivalent positions, it was shown that the binding area as a whole has a larger affinity towards the aromatic residues. Substitution of a D-residue for an L-residue reduced kcat values rather than K m. In addition a remarkable affinity for the urethane-grouping located specifically at subsite S3 was found. A methyloxycarbonyl or benzyloxy — carbonyl group occupying this subsite caused a 5-fold increase in K m as compared with an acetyl or phenylproprionyl group, respectively. Methyloxycarbonyl or benzyloxycarbonyl groups in subsites S2 or S4 showed no such effect.

FDP aldolase has been purified and crystallized from the muscle of codfish. The specific activity of the codfish enzyme was found to be two-thirds that of rabbit muscle aldolase. Other catalytic properties, the molecular weight, and the carboxyl-terminal structure were similar to those of rabbit muscle aldolase. A tryptic peptide containing the Schiff base-forming lysine (1) has been isolated and its primary structure determined as: Ala-Leu-Ser-Asp-His-His-Val-Tyr-Leu-Gln-Gly-Thr-Leu-Leu-βGlys-Pro-Asn-Met-Val-Thr-Ala-Gly-His-Ser-CMCys-Ser-His-Lys. Extensive homology observed in this region of the aldolase molecules isolated from biologically divergent sources indicates the importance of this structure for catalysis of the aldolase reaction.

Fructose diphosphate aldolases have been isolated as crystalline proteins from rat liver, muscle and the Novikoff hepatoma. All three enzymes exhibit identical properties in sedimentation velocity and sedimentation equilibrium ultracentrifugation, corresponding to a molecular weight of 158,000. Dissociation in guanidinium chloride yields subunits of molecular weight approximately 40,000 in each case. Tumor and muscle aldolases exhibit identical electrophoretic properties on cellulose acetate or disc gel which are markedly different from those of liver aldolase. The tumor and muscle enzymes are 50 times more active with fructose-1,6-P2 than with fructose-1-P and treatment of these enzymes with carboxypeptidase A to remove the COOH-terminal tyrosine residues decreases the rate of cleavage of fructose-1,6-p2 so that it becomes equal to the rate of cleavage of fructose-1-P. Native rat liver aldolase, on the other hand, cleaves both substrates at equal rates and digestion with carboxypeptidase, while also liberating tyrosine from the COOH-terminus, does not affect the substrate specificity. The amino acid compositions of muscle and heppatoma aldolases were found to be identical and to differ significantly from that of the rat liver aldolase. Tryptic fingerprints of muscle and tumor aldolases are identical and differ significantly from that of liver aldolase. However, a number of peptides, including the active site peptide, are similar or identical in all three aldolases. The results provide support for the conclusion that the liver aldolase is replaced by muscle aldolase during the development of the Novikoff hepatoma.

The enzyme, l -glutamine d -fructose 6-phosphate amidotransferase (EC 2.6.1.16) of Neurospora crassa , which catalyzes the formation of glucosamine 6-phosphate was shown to be subject to feedback inhibition by uridine diphosphate N -acetyl- d -glucosamine (UDP-GlcNAc). The conclusion is based on the following observations. UDP-GlcNAc, the direct precursor of chitin, did not accumulate in the cell even when its utilization for the synthesis of cell wall chitin was interrupted by the antibiotic polyoxin D, a competitive inhibitor of the chitin synthetase (EC 2.4.1.16). Furthermore, the cellular level of UDP-GlcNAc rose in a short period of time when the amidotransferase was bypassed in vivo by the addition of glucosamine to the growing medium of the fungus. The amidotransferase was purified from N. crassa approximately 85-fold. Kinetic studies showed that UDP-GlcNAc was a potent and specific inhibitor of the amidotransferase, and that it did not alter the Michaelis constant for either l -glutamine or d -fructose 6-phosphate, suggesting that the inhibitor binds at a site on the enzyme distinct from the active site.

Investigations have revealed that sheep kidney pyruvate carboxylase (pyruvate: CO2 ligase (ADP), EC 6.4.1.1) possesses a lysine residue whose integrity is essential for enzymic activity. Modification of the enzyme with amino group reagents such as 1-fluoro-2,4-dinitrobenzene, trinitrobenzene sulphonic acid and potassium cyanate resulted in a loss of catalytic activity. The characterisation of the major radioactive dinitrophenyl-amino acid derivative of 1-fluoro-2,4-dinitro[14C]benzene labelled enzyme as e-dinitrophenyl-lysine confirmed the identity of the reactive residue. Inhibition of the enzyme by 1-fluoro-2,4-dinitrobenzene and trinitrobenzene sulphonic acid was first order with respect to time and inhibitor concentration. From the kinetic data obtained, it was concluded that an average of 1 molecule of these reagents reacts with the enzyme causing inactivation. The rate of inactivation with these reagents was pH dependent, increasing with increasing pH values. The ability of the allosteric effector, acetyl-CoA, to protect the enzyme against inhibition by these reagents suggested that the e-amino group of lysine may be involved in the enzyme-acetyl-CoA interaction. The dissociation constant for the enzyme-acetyl-CoA complex was 5.4·10−4 M.

Data are presented on the sites of covalent binding of acetate and malonate to the soluble pigeon liver fatty acid synthetase. Each of these compounds is bound to each of two peptide fractions which are released from the synthetase by pepsin digestion and then separated by Dowex ion-exchange chromatography. One of these peptide fractions has not been resolved. Further purification and study of the other peptide fraction has resulted in proof of the presence of 4′-phosphopantetheine bound through ester linkage to the peptide chain. In these experiments the peptides were labeled with either acetate-1- 14 C malonate-2- 14 C or pantothenic-1- 14 C acid. Quantitative analysis of the multi-enzyme complex for 4′-phosphopantetheine yielded a value close to 1 mole per mole of synthetase (450,000 gm). The implications of the findings that 1 mole of 4′-phosphopantetheine is found per mole of enzyme and that either acetate or malonate covalently bind to this compound are discussed.

The isolation and sequence of active site peptides from glycogen phosphorylase b is described. These cysteine peptides become exposed to preferential labeling upon removal of pyridoxal 5′-phosphate (PLP) from the enzyme and they have the sequence Ala-Cys, Asx-Ala-Cys-Asp and Asx-Glx-Lys-Cys-Gly-Gly. The first two of these peptides appear to be derived from the PLP binding site of phosphorylase.

In vitro methylation of E. coli tRNAfMet fragments or their reconstituted molecule by rat liver methylase showed that (1) the whole tRNA molecule was not necessary for recognition by tRNA methylase, and (2) the extent and the site of methylation of fragments differed depending upon the type of fragment used. This indicates that the conformational structure of the methyl acceptor molecule is important for recognition by the tRNA methylase.