Restricted active site docking by enzyme-bound substrate enforces the ordered cleavage of prothrombin by prothrombinase.

The preferred pathway for prothrombin activation by prothrombinase involves initial cleavage at Arg(320) to produce meizothrombin, which is then cleaved at Arg(271) to liberate thrombin. Exosite binding drives substrate affinity and is independent of the bond being cleaved. The pathway for cleavage is determined by large differences in V(max) for cleavage at the two sites within intact prothrombin. By fluorescence binding studies in the absence of catalysis, we have assessed the ability of the individual cleavage sites to engage the active site of Xa within prothrombinase at equilibrium. Using a panel of recombinant cleavage site mutants, we show that in intact prothrombin, the Arg(320) site effectively engages the active site in a 1:1 interaction between substrate and enzyme. In contrast, the Arg(271) site binds to the active site poorly in an interaction that is approximately 600-fold weaker. Perceived substrate affinity is independent of active site engagement by either cleavage site. We further show that prior cleavage at the 320 site or the stabilization of the uncleaved zymogen in a proteinase-like state facilitates efficient docking of Arg(271) at the active site of prothrombinase. Therefore, we establish direct relationships between docking of either cleavage site at the active site of the catalyst, the V(max) for cleavage at that site, substrate conformation, and the resulting pathway for prothrombin cleavage. Exosite tethering of the substrate in either the zymogen or proteinase conformation dictates which cleavage site can engage the active site of the catalyst and enforces the sequential cleavage of prothrombin by prothrombinase.

Distribution of amine oxidases and amine dehydrogenases in bacteria grown on primary amines and characterization of the amine oxidase from Klebsiella oxytoca.

The bacteria Klebsiella oxytoca LMD 72.65 (ATCC 8724), Arthrobacter P1 LMD 81.60 (NCIB 11625), Paracoccus versutus LMD 80.62 (ATCC 25364), Escherichia coli W LMD 50.28 (ATCC 9637), E. coli K12 LMD 93.68, Pseudomonas aeruginosa PAO1 LMD 89.1 (ATCC 17933) and Pseudomonas putida LMD 68.20 (ATCC 12633) utilized primary amines as a carbon and energy source, although the range of amines accepted varied from organism to organism. The Gram-negative bacteria K. oxytoca and E. coli as well as the Gram-positive methylotroph Arthrobacter P1 used an oxidase whereas the pseudomonads and the Gram-negative methylotroph Paracoccus versutus used a dehydrogenase for amine oxidation. K. oxytoca utilized several primary amines but showed a preference for those containing a phenyl group moiety. Only a single oxidase was used for oxidation of the amines. After purification, the following characteristics of the enzyme indicated that it belonged to the group of copper-quinoprotein amine oxidase (EC 1.4.3.6): the molecular mass (172,000 Da) of the homodimeric protein; the UV/visible and EPR spectra of isolated and p-nitrophenylhydrazine-inhibited enzyme; the presence and the content of copper and topaquinone (TPQ). The amine oxidase appeared to be soluble and localized in the periplasm, but catalase and NAD-dependent aromatic aldehyde dehydrogenase, enzymes catalysing the conversion of its reaction products, were found in the cytoplasm. From the amino acid sequence of the N-terminal part as well as that of a purified peptide, it appears that K. oxytoca produces a copper-quinoprotein oxidase which is very similar to that found in other Enterobacteriaceae.

Crystal structure of quinohemoprotein amine dehydrogenase from Pseudomonas putida. Identification of a novel quinone cofactor encaged by multiple thioether cross-bridges.

The crystal structure of a quinohemoprotein amine dehydrogenase from Pseudomonas putida has been determined at 1.9-A resolution. The enzyme comprises three non-identical subunits: a four-domain alpha-subunit that harbors a di-heme cytochrome c, a seven-bladed beta-propeller beta-subunit that provides part of the active site, and a small gamma-subunit that contains a novel cross-linked, proteinous quinone cofactor, cysteine tryptophylquinone. More surprisingly, the catalytic gamma-subunit contains three additional chemical cross-links that encage the cysteine tryptophylquinone cofactor, involving a cysteine side chain bridged to either an Asp or Glu residue all in a hitherto unknown thioether bonding with a methylene carbon atom of acidic amino acid side chains. Thus, the structure of the 79-residue gamma-subunit is quite unusual, containing four internal cross-links in such a short polypeptide chain that would otherwise be difficult to fold into a globular structure.