R. Heiner Schirmer

Mitochondrial GTP-AMP Phosphotransferase.

GTP-AMP phosphotransferase has been purified 116-fold with a yield of 24% from beef heart mitochondria using freeze-thawing, alkali and acid treatment and successive column chromatography on phosphocellulose, Sephadex G-100 and blue-dextran--Sepharose. It has crystallized from poly-(ethylene glycol) and is essential homogeneous by sodium dodecylsulfate electrophoresis and isoelectrofocusing. The specific activity of the crystalline preparation was 290 U/mg. The molecular weight was found to be 26000 and the isoelectric point to be 9.8. Amino acid analysis showed 21 aspartic acid or asparagine, 19 threonine, 12 serine, 26 glutamic acid or glutamine, 15 proline, 16 glycine, 14 alanine, 15 valine, 4 methionine, 12 isoleucine, 28 leucine, 7 tyrosine, 7 phenylalanine, 5 histidine, 14 lysine, 16 arginine, 2 tryptophan, no --SS-- bonds or free --SH. Guanosine(5)pentaphospho(5)adenosine is a very strong inhibitor similar to adenosine(5)pentaphospho(5)adenosine as an inhibitor of cytosolic adenylate kinase.

Preparation and characterization of a crystalline human ATP:AMP phosphotransferase

Abstract 1. 1.|An adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) has been purified 100-fold from human muscle with a final yield of 30 mg per kg of muscle. 2. 2.|Single crystals were grown from the electrophoretically homogeneous protein. 3. 3.|The specific activity was found to be 2000 μmoles of ATP produced or consumed per min per mg of protein at 25° and pH 8.0. The Michaelis constants for AMP, ADP, and ATP, respectively, are all in the range of 0.3 mM. 4. 4.|Studies on the physical properties indicate that the protein possesses a sedimentation constant of 2.30 S, a diffusion coefficient of 9.9 · 10 −7 cm 2 · sec −1 , a partial specific volume of 0.74 ml/g and a mol. wt. of 21 500. 5. 5.|Amino acid analysis revealed a total of 194 residues, giving a calculated mol. wt. of 21 700. The composition was determined to be Asx 13 , Thr 13 , Ser 12 , Glx 26 , Pro 7 , Gly 18 , Ala 10 , Val 15 , Met 4 , Leu 17 , Ile 8 , Tyr 7 , Phe 5 , Lys 20 , His 4 , Arg 13 , Trp 0 , Cys 2 , amide ammonia 12 .

The C-terminal fragment of human glutathione reductase contains the postulated catalytic histidine.

The amino acid sequence around the catalytic disulfide in human glutathione reductase is similar to that of other disulfide reductases [I ,3]. Spectroscopic data indicate that intermediate states during catalysis are common to lipoamide dehydrogenase (EC 164.3) and glutathione reductase (EC 1.6.4.2), notably a charge transfer complex between a thiolate ion and the (re)oxidized flavin [4-61. For stabilizing this thiolate, a protonated base was proposed [7,8], in analogy to the ion pair Cys-/His’ in papain ]9,1 O]. By X-ray diffraction analysis of human glutathione reductase, the redox-active disulfide peptide was found to be in contact with the flavin [ 11 ,121. Near Cys-46, the proposed thiolate ion, the sidechain of residue 450 of the other subunit was found in the electron density map. From size and shape it was interpreted as a His 1121. The intention of this study was to sequence the corresponding peptide, to locate it in the electron density map, and to chemically identify residue 450.

Sensitivity of adenylate kinase isozymes from normal and dystrophic human muscle to sulfhydryl reagents

Abstract 1. 1.|Crystalline human muscle adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) possesses two sulfhydryl groups. The enzyme was only partially inactivated when these thiol groups were reacted with mercury or silver compounds. Reaction with Ellmans reagent, however, resulted in complete loss of activity. 2. 2.|The adenylate kinase activity of extracts of normal human muscle was inhibited by Ellmans reagent whereas the adenylate kinase activity of extracts of human liver was insensitive towards all sulfhydryl reagents. 3. 3.|In extracts of diseased human muscle (progressive muscular dystrophy Duchenne type) 40–60% of the adenylate kinase activity remained after treatment with Ellmans reagent. 4. 4.|These and other results indicate that the adenylate kinase isozymes might be an adequate system for investigating the role of SH-proteins in the pathogenesis of muscular dystrophies. 5. 5.|The reaction of mercury compounds with 2-nitro-5-thiobenzoate was used for the quantitative determination of organic mercurials.

Patterns of Folding and Association of Polypeptide Chains

Six levels of structural organization can be distinguished. According to Linderstrom-Lang (176), four levels of structural organization in proteins can be distinguished: primary, secondary, tertiary, and quaternary structure (176). These terms refer to the amino acid sequence, the regular arrangements of the polypeptide backbone, the three-dimensional structure of the globular protein, and the structures of aggregates of globular proteins, respectively. With our present knowledge, two more levels can be added: supersecondary structures denoting physically preferred aggregates of secondary structure and domains referring to those parts of the protein which form well-separated globular regions. An organizational scheme is given in Figure 5–1a. Since renaturation experiments have shown that the amino acid sequence contains the entire structural information (177), the relationship between these levels is dependent, with elements at a lower level determining the elements of higher levels.

Prediction of Secondary Structure from the Amino Acid Sequence

To a rough approximation the secondary structure of a chain segment is a function of the constituent amino acids alone. A glimpse at Dayhoff’s Atlas of Protein Sequence and Structure (20) shows that the number of known amino acid sequences far exceeds the number of known three-dimensional structures. Since the amino acid sequence contains the complete structural information (section 1.2 and Ref. 177), it should be possible to derive the spatial structure from the sequence alone without X-ray analysis. The first step in this direction should be to try to bridge the gap between the two lowest levels of structural organization in proteins (Figure 5–1), between sequence and secondary structure. This organization is approximately hierarchic. But the hierarchy is not strict so that the formation of secondary structure in a given segment of the polypeptide chain does not depend on the sequence in this segment alone. Other segments at a distance along the chain also exert an influence, the functional dependence between the two lowest levels being of a rather nonlocal nature.1

Noncovalent Forces Determining Protein Structure

Noncovalent forces were discovered by van der Waals (1873) in an attempt to explain the deviation of a real gas from the ideal gas law. They are of utmost importance for biological organisms. In particular, they drive the spontaneous folding of polypeptide and nucleic acid chains and the spontaneous formation of membranes. They mediate the mutual recognition of complementary molecular surfaces (“lock and key system” (44)).

Structural Implications of the Peptide Bond

Polypeptides are formed in a series of highly controlled reactions. Amino acids are polymerized into a polypeptide chain on ribosomes in the cell. Polymerization is based on the formation of amide bonds which are usually called “peptide bonds.” The chain direction is defined as pointing from the amino end (N-terminus) to the carboxyl end (C-terminus) as shown in Figure 2–2. This definition coincides with the direction of chain synthesis in vivo, which in turn corresponds to the 5′→3′-direction on the messenger RNA.

The Covalent Structure of Proteins

In section 1.1, we started out with the generalization that the functional form of a protein is obtained merely by synthesizing the polypeptide chain on ribosomes and by letting it fold spontaneously. Although this is the basic concept, there are frequent biological amendments to this scheme which are of great importance. These phenomena are discussed here.

Protein-Ligand Interactions

Proteins are selective in their interactions with cell constituents. In contrast to naturally occurring proteins, chemically synthesized polypeptides of random sequence behave like small children; they touch, bind, and break many low molecular weight metabolites (614). Natural proteins were educated by evolution to touch only a small selection of molecules (586). This was only possible because they learned to form defined compact structures in contrast to synthetic polypeptides. Specific binding is an individual property of individual proteins. Nonbinding, rather than binding, is the result of organized protein structures.