Alignment of the amino terminal amino acid sequence of human cytochrome c oxidase subunits I and II with the sequence of their putative mRNAs

Thirteen of the first fifteen amino acids from the NH2-terminus of the primary sequence of human cytochrome c oxidase subunit I and eleven of the first twelve amino acids of subunit II have been identified by microsequencing procedures. These sequences have been compared with the recently determined 5'-end proximal sequences of the HeLa cell mitochondrial mRNAS and unambiguously aligned with two of them. This alignment has allowed the identification of the putative mRNA for subunit I, and has shown that the initiator codon for this subunit is only three nucleotides away from the 5'-end of its mRNA; furthermore, the results have substantiated the idea that the translation of human cytochrome c oxidase subunit II starts directly at the 5'-end of its putative mRNA, as had been previously inferred on the basis of the sequence homology of human mitochondrial DNA with the primary sequence of the bovine subunit

Isolation and amino acid sequence analysis of a 4,000-dalton dynorphin from porcine pituitary

A 4,000-dalton dynorphin was isolated from porcine pituitary. It has 32 amino acids (Mr = 3,986), with the previously described heptadecapeptide (now called dynorphin A) at its amino terminus and a related tridecapeptide, dynorphin B, at its carboxyl terminus. The two peptides are separated by the "processing signal" Lys-Arg

Ionization behavior of the histidine residue in the catalytic triad of serine proteases

α-Lytic protease is a homologue of the mammalian serine proteases such as trypsin, chymotrypsin, and elastase, and its single histidine residue belongs to the Asp-His-Ser catalytic triad. This single histidine residue has been selectively enriched in the C-2 carbon with 13C. Magnetic resonance studies of the chemical shift and coupling constant (1Jch) behavior of this nucleus as a function of pH suggest that the imidazole ring is neutral above pH 5 and therefore that the group which is known to ionize with pKa near 6.7 must be the aspartic acid residue. Implications of these new pKa assignments for the catalytic mechanism of serine proteases are discussed and include the absence of any need to separate charge during catalysis. The histidine residue plays two roles. (a) It insulates the aspartic acid from an aqueous environment and accordingly raises its pKa. (b) It serves as a bidentate base to accept a proton from the serine at one of its nitrogens and concertedly transfer a proton from its other nitrogen to the buried carboxylate anion during formation of the tetrahedral intermediate

A gas-liquid solid phase peptide and protein sequenator

A new miniaturized protein and peptide sequenator has been constructed which uses gas phase reagents at the coupling and cleavage steps of the Edman degradation. The sample is embedded in a matrix of Polybrene dried onto a porous glass fiber disc located in a small cartridge-style reaction cell. The protein or peptide, though not covalently attached to the support, is essentially immobile throughout the degradative cycle, since only relatively apolar, liquid phase solvents pass through the cell. This instrument can give useful sequence data on as little as 5 pmol or protein, can perform extended sequence runs (greater than 30 residues) on subnanomole quantities of proteins purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and can sequence hydrophobic peptides to completion. The sequenator is characterized by a high repetitive yield during the degradation, low reagent consumption, low maintenance requirements, and a degradative cycle time of only 50 min using a complete double cleavage program

Improved assembly and variant detection of a haploid human genome using single-molecule, high-fidelity long reads

The sequence and assembly of human genomes using long-read sequencing technologies has revolutionized our understanding of structural variation and genome organization. We compared the accuracy, continuity, and gene annotation of genome assemblies generated from either high-fidelity (HiFi) or continuous long-read (CLR) datasets from the same complete hydatidiform mole human genome. We find that the HiFi sequence data assemble an additional 10% of duplicated regions and more accurately represent the structure of tandem repeats, as validated with orthogonal analyses. As a result, an additional 5 Mbp of pericentromeric sequences are recovered in the HiFi assembly, resulting in a 2.5-fold increase in the NG50 within 1 Mbp of the centromere (HiFi 480.6 kbp, CLR 191.5 kbp). Additionally, the HiFi genome assembly was generated in significantly less time with fewer computational resources than the CLR assembly. Although the HiFi assembly has significantly improved continuity and accuracy in many complex regions of the genome, it still falls short of the assembly of centromeric DNA and the largest regions of segmental duplication using existing assemblers. Despite these shortcomings, our results suggest that HiFi may be the most effective standalone technology for de novo assembly of human genomes

Nuclear magnetic resonance studies of the catalytic mechanism of proteolytic enzymes. Ionization behavior of the histidine residue in the catalytic triad of alpha-lytic protease--implications for the catalytic mechanism of serine proteases. Ionization behavior of enzymic and inhibitor groups in the tetrahedral adduct between alpha-lytic protease and a peptide aldehyde. Kinetics of pepsin-catalyzed hydrolysis of N-tri-fluoroacetyl amino acids

NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document. PART I Selective [superscript 13]C enrichment of C-2 of the single histidine residue of the serine protease [alpha]-lytic protease has allowed direct study of the Asp-His-Ser catalytic triad by magnetic resonance techniques. Both the chemical shift of C-2 and the coupling between C-2 and its directly bonded hydrogen have been observed as a function of pH. The results indicate that only below pH 3.3 does the histidine imidazole ring become protonated and only above pH 6.7 does the aspartic acid residue lose a proton to generate a carboxylate anion. Over the pH range 3.3-6.7, the catalytic triad contains a neutral aspartic acid and neutral histidine residue--not the ionized forms hitherto assumed. This interpretation of the ionization characteristics of the catalytic triad leads to a proposed catalytic mechanism which avoids any requirement for unfavorable charge separation in the transition state. The histidine residue plays two roles: (i) it provides insulation between water and the buried carboxylate anion, thus ensuring the latter a hydrophobic environment, and (ii) it provides a relay for net proton transfer from serine hydroxyl to carboxylate anion. The aspartate anion acts as the ultimate base which holds a proton during catalysis. An anionic, rather than a neutral, base both avoids the necessity of charge separation and, by giving the catalytic locus an overall negative charge, assists preferential expulsion of product relative to substrate from the active site. Relaxation measurements (T[subscript 1], T[subscript 2], and nuclear Overhauser enhancement) indicate that, over the pH range of enzymic activity, the histidine residue is held rigidly within the protein. PART II Magnetic resonance techniques have been used to study ionization behavior of enzymic and inhibitor moieties in the tetrahedral adduct (hemiacetal) formed between [alpha]-lytic protease and a peptide aldehyde, N-Ac-L-Ala-L-Pro-L-alaninal. Chemical shift, coupling constant, and relaxation measurements of [superscript 13]C-enriched C-2 of the catalytic histidine residue indicate that at pH > 6.25 the complex contains neutral aspartic acid, neutral histidine, and negatively charged inhibitor. Below pH 6.25, both the inhibitor oxyanion and the histidine become protonated in a cooperative ionization process which forces the histidine from its rigidly-held position as a member of the catalytic triad into a solution-like environment. This behavior by a complex thought to resemble the transition state for serine protease-catalyzed hydrolysis of ester and amide substrates supports proposals for a catalytic mechanism which involves a minimum of charge separation in the transition state. It also attests to the power of the intricate hydrogen-bonding network (previously observed in x-ray diffraction studies) to stabilize an otherwise high-energy intermediate and thereby achieve catalysis. PART III The acidic gastric proteases, pepsin and gastricsin, have been found to catalyze hydrolysis of several N-trifluoroacetyl-L-amino acids with aromatic side chains. This catalytic activity is lost when they are chemically modified so as to inactivate their proteolytic activity. Magnetic resonance techniques were used to follow the porcine pepsin-catalyzed hydrolysis of N-trifluoroacetyl L-phenylalanine in the pH range 1.7-5.4. This study revealed that non-productive binding strongly influences the observed kinetic parameters and that productive enzyme-substrate binding requires an anionic substrate (pK[subscript a] 2.8) and an undissociated group (pK[subscript a] 3.7) on the free enzyme. Binding is also affected by ionization of a group on the free enzyme with a pK[subscript a] near 4.8. A kinetic isotope effect [...] has been observed for the reaction which suggests that proton transfer is involved in the rate-limiting step. A new mechanism--one involving three carboxylic acid groups on the enzyme and an intermediate in which the amino moiety is noncovalently held by the enzyme after release of the acyl moiety--is proposed to explain these and previous observations on catalysis by pepsin

Protein Sequence Analysis: Automated Microsequencing

The automated microsequencing of proteins can now be carried out at the 5- to 10-picomoles (submicrogram) level on polypeptides obtained directly from one- and two-dimensional gel electrophoresis. The techniques are applicable to polypeptides ranging in size from small peptides (less than 10 residues) to large proteins (more than 1000 residues)

New Protein Sequenator with Increased Sensitivity

New developments in automated amino acid sequence analysis permit a 10^4-fold increase in sensitivity (to ~ 10-picomole level) over that initially described by Per Edman in 1967

Human Platelet-Derived Growth Factor (PDGF): Amino-Terminal Amino Acid Sequence

Human platelet-derived growth factor (PDGF) obtained from outdated human platelets was subjected to amino-terminal amino acid sequence analysis by automated Edman degradation. Despite the apparent presence of limited proteolytic degradation of the protein derived from this method, the sequence analysis reveals two primary peptide sequences and suggests that active PDGF is composed of two, possibly homologous, peptides linked by a disulfide bond or bonds

Mechanism of action of serine proteases: tetrahedral intermediate and concerted proton transfer

Stopped-flow spectrophotometry and proton inventory experiments have been used to define the reaction pathway for hydrolysis of a specific peptide substrate, Ac-L-Ala-L-Pro-L-Ala p-nitroanilide, by the serine proteases elastase and α-lytic protease. The stopped-flow studies reveal the existence and buildup of a tetrahedral adduct between the active site serine hydroxyl group and the sensitive carbonyl group of the substrate. The decomposition of this tetrahedral intermediate to the acyl enzyme and p-nitroaniline is the rate-limiting step for the hydrolytic reaction. The proton inventory data suggest the simultaneous transfer of two protons (presumably from the catalytic carboxyl of Asp-102 to Nπ of the catalytic imidazole of His-57 and from Nτ of the imidazole to the anilide NH) in the transition state leading to breakdown of the tetrahedral complex. That these proton transfers occur in a concerted, rather than stepwise, process attests to the ability of enzymes to lower the enthalpy of activation most effectively when the precise alignment of a highly specific substrate and catalytic groups minimizes the entropy of activation