Molecular chaperones of the Hsp70 type transiently sequester unfolded segments of proteins and promote their correct folding. Target peptides were labeled with an environmentally sensitive fluorophore so that their binding to the molecular chaperone DnaK of Escherichia coli could be followed in real time. The two-step process was characterized by relaxation times of 27 seconds and 200 seconds with 2 microM DnaK and 0.1 microM ligand at 25 degrees C. In the presence of adenosine triphosphate, the formation of the complex was greatly accelerated and appeared to be a single-exponential process with a relaxation time of 0.4 second. The binding-release cycle of DnaK thus occurs in the time range of polypeptide chain elongation and folding and is too fast to be stoichiometrically coupled to the adenosine triphosphatase activity of the chaperone (turnover number, 0.13 per minute at 30 degrees C).

A total of 150 amino acid sequences of vitamin €3,-dependent enzymes are known to date, the largest contingent being furnished by the aminotransferases with 51 sequences of 14 different enzymes. All aminotransferase sequences were aligned by using algorithms for sequence comparison, hydropathy patterns and secondary structure predictions. The aminotransferases could be divided into four subgroups on the basis of their mutual structural relatedness. Subgroup I comprises aspartate, alanine, tyrosine, histidinol-phosphate, and phenylalanine aminotransferases ; subgroup I1 acetylornithine, ornithine, o-amino acid, 4-aminobutyrate and diaminopelargonate aminotransferases ; subgroup 111 D-alanine and branched-chain amino acid aminotransferases, and subgroup IV serine and phosphoserine aminotransferases. (N-1) Profile analysis, a more stringent application of profile analysis [Gribskov, M., McLachlan, A. D. and Eisenberg, D. (1987) Proc. Nut1 Acud. Sci. USA 84, 4355-43581, established the homology among the enzymes of each subgroup as well as among all subgroups except subgroup 111. However, similarity of active-site segments and the hydropathy patterns around invariant residues suggest that subgroup 111, though most distantly related, might also be homologous with the other aminotransferases. On the basis of the comprehensive alignment, a new numbering of amino acid residues applicable to aminotransferases (AT) in general is proposed. In the multiply aligned sequences, only four out of a total of about 400 amino acid residues proved invariant in all 51 sequences, i.e. Gly(314AT)197, Asp/Glu(340AT)222, Lys(385AT)258 and Arg(562AT)386, the number not in parentheses corresponding to the structure of porcine cytosolic aspartate aminotransferase. Apparently, the aminotransferases constitute a group of homologous proteins which diverged into subgroups and, with some exceptions, into substrate-specific individual enzymes already in the universal ancestor cell.The aminotransferases catalyze the reversible transfer of amino groups from amino acids to 0x0 acids, one of the numerous transformations of amino acids that are performed by vitamin-B,-dependent enzymes. Among the various groups of pyridoxal-5'-phosphate-dependent enzymes, the aminotransferases excel by the highest number of reported amino acid sequences, i.e. one third of the total of about 150 sequences of pyridoxal-5'-phosphate-dependent enzymes known to date. The homology of aspartate, tyrosine and histidinol-phosphate aminotransferases has already been reported. On alignment of 16 amino acid sequences of these enzymes, 12 amino acid residues, mainly ligands of the coenzyme or the substrate, proved invariant out of a total of about 400 residues (Mehta et al., 1989). Since the publication of that report, the number of known aminotransferase sequences has doubled.In a continuation of our studies on the evolutionary relationships among vitamin B,-dependent enzymes, all known sequences of aminotransferases were aligned on the basis of sequence similarity, hydropathy pa...

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Pyridoxal-5'-phosphate-dependent enzymes catalyze manifold reactions in the metabolism of amino acids. A comprehensive comparison of amino acid sequences has shown that most of these enzymes can be assigned to one of three different families of homologous proteins. The sequences of the enzymes of each family were aligned and their homology confirmed by profile analysis. Scrutiny of the reactions catalyzed by the enzymes showed that their affiliation with one of the three structurally defined families correlates in most cases with their regio-specificity.In the largest family, the covalency changes of the substrate occur at the same carbon atom that carries the amino group forming the irnine linkage with the coenzyme. This family was thus named a family. It comprises glycine hydroxymethyltransferase, glycine C-acetyltransferase, 5-aminulevulinate synthase, 8-amino-7-oxononanoate synthase, all aminotransferases (with the possible exception of subgroup 111), a number of other enzymes relatively closely related with the aminotransferases and very likely a certain group of amino acid decarboxylases as well as tryptophanase and tyrosine phenol-lyase which, however, catalyze p-elimination reactions. The p family includes L-and Dserine dehydratase, threonine dehydratase, the /? subunit of tryptophan synthase, threonine synthase and cysteine synthase. These enzymes catalyze p-replacement or p-elimination reactions. The y family incorporates 0-succinylhomoserine (thio1)-lyase, 0-acetylhomoserine (thio1)-lyase, and cystathionine y-lyase, which catalyze y-replacement or y-elimination reactions, as well as cystathionine P-lyase.The a and y family might be distantly related with one another, but are clearly not homologous with the / 3 family. Apparently, the primordial pyridoxal-5'-phosphate-dependent enzymes were regio-specific catalysts, which first specialized for reaction specificity and then for substrate specificity. The following pyridoxal-5'-phosphate-dependent enzymes seem to be unrelated with the a, /? or y family by the criterion of profile analysis: alanine racemase, selenocysteine synthase, and many amino acid decarboxylases. These enzymes may represent yet other families of B, enzymes.Pyridoxal-5'-phosphate-dependent enzymes (B, enzymes) catalyze such a wide variety of transformations of amino acids that they are found in no fewer than four out of the total six EC classes of enzymes (Enzyme Nomenclature, 1992). This paper is part of a study on the evolutionary relationships among these multifarious enzymes. Previously, we have found that most if not all aminotransferases constitute a group of homologous proteins . Profile analysis, an algorithm for the detection of distant relationships between amino acid sequences (Gribskov et al., 1990), showed, however, that the evolutionary relationships extend beyond the group of aminotransferases and include other B, enzymes Mehta and Christen, 1994). This study compares the currently known amino acid sequences of B, enzymes and shows that many of them belong to one of th...

Hsp70-Hsp40-NEF and possibly Hsp100 are the only known molecular chaperones that can use the energy of ATP to convert stably pre-aggregated polypeptides into natively refolded proteins. However, the kinetic parameters and ATP costs have remained elusive because refolding reactions have only been successful with a molar excess of chaperones over their polypeptide substrates. Here we describe a stable, misfolded luciferase species that can be efficiently renatured by substoichiometric amounts of bacterial Hsp70-Hsp40-NEF. The reactivation rates increased with substrate concentration and followed saturation kinetics, thus allowing the determination of apparent V(max)' and K(m)' values for a chaperone-mediated renaturation reaction for the first time. Under the in vitro conditions used, one Hsp70 molecule consumed five ATPs to effectively unfold a single misfolded protein into an intermediate that, upon chaperone dissociation, spontaneously refolded to the native state, a process with an ATP cost a thousand times lower than expected for protein degradation and resynthesis.