Alkyl isocyanates as active site-specific inhibitors of chymotrypsin and elastase.

Alkyl isocyanates react specifically with the two serine proteinases, chymotrypsin and elastase, to yield inactive enzyme derivatives containing 1 male of reagent per mole of enzyme. Octyl isocyanate inactivates chymotrypsin only, while butyl isocyanate inactivates both enzymes but shows greater efficiency toward elastase than toward chymotrypsin. These reagents may thus represent unique chemical "yardsticks" for the measurement of the relative dimensions of the active sites of the two very similar enzymes.

Posttranslational covalent modification of proteins.

A search for derivatized amino acids in proteins has shown that the extent of posttranslational modification of proteins is quite substantial. While only 20 primary amino acids are specified in the genetic code and are involved as monomer building blocks in the assembly of the polypeptide chain, about 140 amino acids and amino acid derivatives have been identified as constituents of different proteins in different organisms. A brief consideration of the questions about where and when the derivatization reactions occur, how the specificity of the reactions is established, and how the posttranslational modifications can facilitate biological processes, reveal a need for more information on all these points. Answers to these questions should represent significant contributions to our understanding of biochemistry and cell biology.

Post-translational modifications of proteins: some problems left to solve.

Three major questions regarding the post-translational modification of amino acid side chains in proteins are briefly considered: (1) What are the biological functions of the reactions, (2) what is the specificity of the processing reactions in selecting only a few or sometimes even only one residue for modification, and (3) how do we solve the uniqueness of the processing steps in the production of recombinant proteins? The answers to these questions are not obvious at this time.

Specificity determinants of acylaminoacyl-peptide hydrolase.

In an attempt to explore how specific features of the substrate's primary structure may affect the activity of rabbit muscle acylaminoacyl-peptide hydrolase (EC 3.4.19.1), a number of acetylated peptides containing specific amino acid replacements in specific positions were prepared and compared as substrates for the hydrolase. The principal variants were D-Ala, Pro, and positive charges (His, Arg, Lys); in addition, the effect of the length of the peptide was also investigated in a less systematic manner. The substrates were either prepared by direct acetylation of peptides, by extension of the N-terminus with acetylamino acids or acetylpeptides, activated as N-hydroxysuccinimide esters, or by isolation of the N-terminal peptides from naturally occurring acetylated proteins. It was found that D-Ala on either side of the bond to be cleaved (positions 1 and 2) completely inhibited the enzymatic activity, whereas acetylated peptides with D-Ala in positions 3 or 4 were as good substrates as those containing L-Ala. Peptides with Pro in positions 2 were also inactive, and most of the peptides with Pro in the third position were very poor substrates; only the peptide Ac-AAP gave reasonably high activity (30% of Ac-AAA), which was reduced to 1-2% if additional residues were present at the C-terminus (Ac-AAPA, Ac-AAPAA). The presence of a positive charge in positions 2, 3, 4, 5, and 6 gave strong reduction in hydrolase activity varying with the charge's distance from the N-terminus from 0 to 15-20% of the rates obtained with the reference peptides without positive charges.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the specificity of acetylaminoacyl-peptide hydrolase.

In a continuing attempt to explore the types of specificity determinants that may affect protein-protein (peptide) interactions, a number of short (2-5 residues) acetylated peptides have been compared as substrates for the enzyme acetylaminoacyl-peptide hydrolase (EC 3.4.19.1). The reference substrate was Ac-AAAA, and most of the other substrates were derived from this basic structure by single amino acid substitutions. The Km and kcat for the different substrates were determined by standard steady-state kinetics, and the corresponding delta delta GT++ value derived from kcat/Km was used for the comparison, setting delta detal GT++ for Ac-AAAA equal to 0. The best substrates were found to be those containing negative charges (Asp > Glu) or aromatic residues in positions 1', 2', or 3' (delta delta GT++ values of 2-5 kJ); the negative charge provided by the C-terminus of the substrate also appears to be important, since the amide and O-Me ester derivatives caused a change in delta delta GT++ values of -7 to -8 kJ from the reference peptide. The stimulating effect of the negative charges is consistent with the inhibitory effect of positive charges in similar peptides (Krishna RG, Wold F, 1992, Protein Sci 1:582-589), and the proposed active site model incorporates subsites for both charge-charge and hydrophobic interactions. In assessing all the data, it is clear that the properties of the individual substrates reflect the total make-up of each peptide and not only the effect of a single residue in a given position.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on N alpha-acylated proteins: the N-terminal sequences of two muscle enolases.

A standard procedure for the identification of the N-terminal amino acid in N alpha-acylated proteins has been developed. After exhaustive proteolysis, the amino acids with blocked alpha-amino groups are separated from positively charged, free amino acids by ion exchange chromatography and subjected to digestion with acylase I. Amino acid analysis before and after the acylase treatment identifies the blocked N-terminal amino acid. A survey of acylamino acid substrates showed that acylase will liberate all the common amino acids except Asp, Cys or Pro from their N-acetyl-and N-butyryl derivatives, and will also catalyze the hydrolysis of N-formyl-Met and N-myristyl-Val. Thus, the procedure cannot identify acylated Asp, Cys or Pro, nor, because of the ion exchange step, N alpha-acyl-derivatives of Arg, Lys or His. Whenever the protease treatment releases free acylamino acids, the remaining amino acids should be detected. When applied to several proteins, the procedure confirmed known N-terminal acylamino acids and identified acyl-Ser in enolases from chum and coho salmon muscle and in pyruvate kinase from rabbit muscle, and acyl-Thr in phosphofructokinase from rabbit muscle. The protease-acylase assay has been used to identify blocked peptides from CNBr- or protease-treated proteins. When such peptides were treated with 1 N HCl at 110 degrees for 10 min, sufficient yields of deacylated, mostly intact, peptide were obtained to permit direct automatic sequencing. The N-terminal sequences of rabbit muscle and coho salmon enolase were determined in this way and are compared to each other and to the sequence of yeast enolase.

Specificity studies of the GDP-[L]-fucose: 2-acetamido-2-deoxy-beta-[D]-glucoside (Fuc-->Asn-linked GlcNAc) 6-alpha-[L]-fucosyltransferase from rat-liver Golgi membranes.

The specificity of Golgi-membrane glycoprotein 6-alpha-[L]-fucosyltransferase [GDP-[L]-fucose: 2-acetamido-2-deoxy- beta-[D]-glucoside (Fuc-->Asn-linked GlcNAc) 6-alpha-[L]-fucosyltransferase; EC 2.4.1.68] has been assessed with regard to substrate covalent structures and the effect of a protein matrix on the conformational display of those covalent structures. Specificity was studied by direct comparison of the substrate quality of nine 6-biotinamidohexanoylAsn (= R) derivatives of intermediates and products in the pathway from Man5GlcNAc2-R to a fully sialylated biantennary complex-type glycan. The Man5 derivative and the sialic acid-containing glycans were completely inactive as substrates. The other glycans were all fucosylated; the best substrate was GlcNAcMan3GlcNAc2-R. The protein-matrix effect was studied by comparing the substrate quality of the same 6-biotinamidohexanoylAsn derivatives as well as the corresponding biotinylAsn derivatives free in solution and bound to streptavidin. On the basis of a model derived from the known 3D structure of biotin (biocytin)-saturated streptavidin, it was predicted that the fucosylation site in the substrates would be completely masked in the biotin-binding pocket in the biotinyl derivatives (proximal display), and at least partially masked in the 6-biotinamidohexanoyl derivatives (distal display). The activity measurements were in agreement with these predictions; the glycan structures GlcNAcMan5GlcNAc2-, GlcNAcMan3GlcNAc2-, and GlcNAc2-Man3GlcNAc2- were readily fucosylated as derivatives free in solution, but were totally inert in the proximal complex with streptavidin. In the distal complexes the latter two structures were found to be fucosylated very slowly while the former structure was inactive.

Characterization of gamma-glutamyl transpeptidase from the Rathke's gland secretions of Kemp's ridley sea turtles (Lepidochelys kempi).

The secretion produced by Rathke's glands of Kemp's ridley sea turtles (Lepidochelys kempi) contains the enzyme gamma-glutamyl transpeptidase. The approximately 200 kDa enzyme contains two different subunits, alpha (54 kDa) and beta (21 kDa), in an unknown stoichiometry. The enzyme transfers gamma-Glu from a number of different donors, such as glutamine, glutathione, S-Me-glutathione, N epsilon(gamma-Glu)-Lys, gamma-Glu-Ala, and other gamma-glutamyl amino acids, either to water or to a variety of acceptor substrates. It appears that a free alpha-amino group is the preferred acceptor. The enzyme is not inhibited by typical sulfhydryl reagents such as N-ethyl-maleimide, p-(chloro)mercuri-benzoate or 5,5'-dithio-bis-(2-nitrobenzoate) or by the active Ser reagent tosyl fluoride. Maleate stimulates the activity of the enzyme, and in the presence of 100 mM maleate 2 mM tosyl fluoride becomes an inactivator of the enzyme. The catalytic and molecular properties of the turtle gamma-glutamyl transpeptidase are similar to those established for mammalian gamma-glutamyl transpeptidase. Neither the physiological role of the enzyme nor the biological function of the secretion in which it occurs is understood at this time.

Introduction of artificial crosslinks into proteins.

In this chapter, we present a brief overview of the current status of protein crosslinking technology. An attempt is made to compare the natural crosslinks and natural crosslinking agents to the artificial ones, and a brief section is devoted to the potential use of enzymes (transglutaminase and peroxidase) as crosslinking agents in vitro. Homobifunctional (x-R-x) and heterobifunctional (x-R-y) reagents are considered in terms of the kinds of functional groups and R-groups that have been used in protein crosslinking, and some examples of reagents and applications from the recent literature are tabulated.