Crystal structures of seryl-tRNA synthetase from Thermus thermophilus complexed with two different analogs of seryl adenylate have been determined at 2.5 A resolution. The first complex is between the enzyme and seryl-hydroxamate-AMP (adenosine monophosphate), produced enzymatically in the crystal from adenosine triphosphate (ATP) and serine hydroxamate, and the second is with a synthetic analog of seryl adenylate (5'-O-[N-(L-seryl)-sulfamoyl]adenosine), which is a strong inhibitor of the enzyme. Both molecules are bound in a similar fashion by a network of hydrogen bond interactions in a deep hydrophilic cleft formed by the antiparallel beta sheet and surrounding loops of the synthetase catalytic domain. Four regions in the primary sequence are involved in the interactions, including the motif 2 and 3 regions of class 2 synthetases. Apart from the specific recognition of the serine side chain, the interactions are likely to be similar in all class 2 synthetases.

The primary structure ofglucoamylase G1 (EC 3.2.1.3) from Aspergillus niger has been determined. Fragments of GI were obtained by cleavage with either cyanogen bromide, hydroxylamine, or S. aureus V8 protease. The resulting peptides were separated using ion exchange chromatography on DEAE-Sephacel, gel filtration, and affinity chromatography on Con A-Sepharose. Secondary fragments were generated by cleavage with either o-iodosobenzoic acid or BNPS-skatole as well as by digestion with S. aureus V8 protease, trypsin, and endoproteinase Lys-C. These peptides were purified by the procedures mentioned above and by reverse phase HPLC. The present fragments were amino acid sequenced and this permitted, in combination with the tryptic peptides (Carlsberg Res. Commun. 48, 517-527 (1983)), identification of 574 of the 614 amino acid residues in Gl. Sequencing of glucoamylase Gl cDNA, constructed from A. niger total mRNA, enabled deduction of the sequence of the remaining 40 amino acid residues localized to 6 short stretches. From the alignment of the fragments the complete primary structure of the enzyme was established. The amino acid sequence corresponds to a molecular weight of the polypeptide moiety of 65,424. Including both hexosamine and neutral carbohydrate contents the molecular weight of the present sample of G1 was calculated to be about 82,000.The majority of the carbohydrate of Gl is found in a highly glycosylated region of 70 amino acid residues which comprises about 35 O-glycosyl serine and threonine residues. This region ends approximately 100 residues from the C-terminus of the enzyme. Two N-glycosylated positions were found in the central part of the polypeptide chain. The molecule contains 9 half-cystine residues. No homology is apparent between the sequence of glycoamylase and various at-amylases.Abbreviations: BNPS-skatole = 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine; ca = citraconyl; Con A = concanavalin A; DFP = diisopropylfluorophosphate; DPCC = diphenylcarbamyl chloride; EDTA = ethylenediaminetetraacetic acid, disodium salt; Hepes = N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid; Nemac = N-ethylmorpholine acetate; HPLC = high pressure liquid chromatography; PTH = phenylthiohydantoin; SDS-PAGE = polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate; 2-pe = 2-pyridylethyl; G I designates the larger and G2 the smaller of the forms ofglucoamylase from A. niger (44).

Human factor VIII has been isolated from a high purity factor VIII concentrate by immunoaffmity chromatography and HPLC on Mono Q gel. Two fractions of factor VIII were obtained with a specific activity of w7000 units/mg.The major fraction contained eight peptide chains of 200, 180, 160, 150, 135, 130, 115, and 105 kDa plus one doublet chain of 80 kDa. The minor fraction contained one peptide chain of 90 kDa plus the chain of 80 kDa. Both fractions were activated by thrombin to the same extent. Amino-terminal amino acid sequence analysis was performed on the 180-kDa, 130-kDa, and 90-kDa chains and showed an identical amino-terminal sequence in these chains. Each chain from 200 kDa to 90 kDa was linked to one 80-kDa chain by a metal-ion bridge(s).Studies on factor VIII in plasma and cryoprecipitate, prepared and gel ifitered in the presence of protease inhibitors, showed that one 200-kDa plus one 80-kDa chain were the only or dominating chains in the materials and may represent native factor VIII. The results indicated that all chains from 180 kDa to 90 kDa are fragments of the 200-kDa chain. All of these more or less fragmented chains form active factor VIII complexes with the 80-kDa chain.Factor VIII (antihemophilic factor) is the protein that is deficient or absent in individuals with classic hemophilia, an X-chromosome-linked bleeding disorder. It participates in the intrinsic pathway of blood coagulation as a cofactor in the activation of factor X by factor IXa, in the presence of phospholipid and calcium (1-7). In plasma, factor VIII is noncovalently bound to von Willebrand factor (vWF), a high molecular weight protein involved in primary hemostasis. Due to the low concentration of factor VIII in plasma and to the fact that factor VIII is highly susceptible to degradation by serine proteases, it is only recently that highly purified factor VIII, free of vWF, has been obtained. Bovine factor VIII, as isolated from plasma, is composed of three peptide chains of 93 kDa, 88 kDa, and 85 kDa (8). Factor VIII purified from porcine plasma has been shown to consist of subunits of 160 kDa, 130 kDa, 82 kDa, and 76 kDa (9). Purified human factor VIII has been prepared from commercial factor VIII concentrate and had a specific activity of 2294 units/mg (10). NaDodSO4/PAGE showed several peptide chains having molecular sizes of 188-79 kDa. The majority of the material was related to factor VIII as shown by immunoblot technique with use of monoclonal antibodies against factor VIII (11). A preparation of human factor VIII from cryoprecipitate, in the presence of seine protease inhibitors, has also been reported with the specific activity of 4740 units/mg (12). Unreduced NaDodSO4/PAGE showed predominant bands at 360 kDa, 210 kDa, and 90 kDa and an 80 kDa/79 kDa doublet band.Immunoblotting using monoclonal antibodies suggested that the 360 kDa component was a precursor to the other components. Thus different results are obtained when different starting materials and procedures for purification are used.The human fac...

The almost universal appreciation for the importance of zinc in metabolism has been offset by the considerable uncertainty regarding the proteins that store and distribute cellular zinc. We propose that some zinc proteins with so-called zinc cluster motifs have a central role in zinc distribution, since they exhibit the rather exquisite properties of binding zinc tightly while remaining remarkably reactive as zinc donors. We have used zinc isotope exchange both to probe the coordination dynamics of zinc clusters in metallothionein, the small protein that has the highest known zinc content, and to investigate the potential function of zinc clusters in cellular zinc distribution. When mixed and incubated, metallothionein isoproteins-1 and -2 rapidly exchange zinc, as demonstrated by fast chromatographic separation and radiometric analysis. Exchange kinetics exhibit two distinct phases (k fast Ӎ 5000 min ؊1 ⅐M ؊1 ; k slow Ӎ 200 min ؊1 ⅐M ؊1 , pH 8.6, 25؇C) that are thought to ref lect exchange between the three-zinc clusters and between the four-zinc clusters, respectively. Moreover, we have observed and examined zinc exchange between metallothionein-2 and the Gal4 protein (k Ӎ 800 min ؊1 ⅐M ؊1 , pH 8.0, 25؇C), which is a prototype of transcription factors with a two-zinc cluster. This reaction constitutes the first experimental example of intermolecular zinc exchange between heterologous proteins. Such kinetic reactivity distinguishes zinc in biological clusters from zinc in the coordination environment of zinc enzymes, where the metal does not exchange over several days with free zinc in solution. The molecular organization of these clusters allows zinc exchange to proceed through a ligand exchange mechanism, involving molecular contact between the reactants.The biological coordination chemistry of zinc is particularly rich in structural motifs (1), apparently reflecting the numerous functions and general importance of this element in biology (2). One particular motif is that found in biological zinc clusters (3), polynuclear complexes in which zinc is coordinated exclusively to thiolate sulfurs of cysteine residues. Their prominent structural feature includes both bridging and terminal cysteine ligands. Each zinc atom resides in a tetrahedral coordination environment and is apparently unable to accommodate additional ligands. How this coordination relates to biological function is unknown. We will address this question with reference to metallothionein (MT), a bilobal protein harboring two of these clusters.Tetranuclear and trinuclear zinc clusters ( Fig. 1 A and B) are located in two separate domains of mammalian MTs (4, 5). The three-metal cluster in the N-terminal ␤-domain has 6 terminal and 3 bridging cysteine ligands, whereas the four-metal cluster in the C-terminal ␣-domain has 11 cysteines, 6 of the terminal type and 5 that form bridges. As a consequence, all 20 cysteines of MT are involved in the binding of seven zinc atoms. Their unparalleled metal͞ligand ratio makes these biological zinc cluste...

Peptide fragments were generated by enzymic or chemical degradation of the small form, G2, and the large form, G1, of Aspergillus niger glucoamylase (EC 3.1.2.3). The G2 form was either identical to residues Ala1– Pro512 or to Ala1– Ala514 of the G1 polypeptide chain containing 616 amino acid residues. Structural analysis of the O‐linked carbohydrates from the 70‐amino‐acid‐residues long extensively glycosylated segment of G2 revealed no significant differences in the contents of single mannose and oligosaccharide units in comparison to the corresponding region of G1. The results suggest that the present G2 form has been generated by limited proteolysis of the larger G1. In contradistinction to this, a recently reported splicing out of an intervening sequence from G1 mRNA leads to a smaller mRNA coding for a G2 protein product with a different COOH‐terminal sequence than the G2 form described in the present work [Boel et al. (1984) EMBO J. 3, 1097–1102].