A 350 amino acid soluble fragment of the intracellular catalytic domain of the human transmembrane leukocyte antigen related (LAR) protein tyrosine phosphatase has been purified 17-fold to greater than 90% purity from an Escherichia coli expression vector in quantities sufficient for kinetic and structural characterization. To assess substrate specificity, phosphotyrosine peptides corresponding to autophosphorylation sites of the two major classes of tyrosine kinases have been synthesized. Thus 6-12-residue phosphotyrosine peptides of the insulin receptor and epidermal growth factor receptor kinase domains and of the autophosphorylation and C-terminal regulatory sites of p60src and p56lck have been analyzed for kcat and KM by using a nonradioactive chromogenic assay for Pi release. The catalytic domain of LAR PTPase shows kcat values of 20-70 s-1 for phosphotyrosine peptides and affinities that vary 150-fold from 27 microM to 4.1 mM.

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The purified nickel-containing CO dehydrogenase complex isolated from methanogenic Methanosarcina thermophila grown on acetate is able to catalyze the exchange of [1-14C] acetyl-coenzyme A (CoA) (carbonyl group) with 12CO as well as the exchange of [3'-32P]CoA with acetyl-CoA. Kinetic parameters for the carbonyl exchange have been determined: Km (acetyl-CoA) = 200 ,uM, Vmax = 15 min-'. CoA is a potent inhibitor of this exchange (K1 = 25 ,uM) and is formed under the assay conditions because of a slow but detectable acetyl-CoA hydrolase activity of the enzyme. Kinetic parameters for both exchanges are compared with those previously determined for the acetyl-CoA synthase/CO dehydrogenase from the acetogenic Clostridium thermoaceticum. Collectively, these results provide evidence for the postulated role of CO dehydrogenase as the key enzyme for acetyl-CoA degradation in acetotrophic bacteria.Acetogens and methanogens possess novel pathways for the synthesis of acetyl-coenzyme A (acetyl-CoA) from single carbon units which allow these organisms to grow on CO2 as the sole carbon source (5). Other strains of methanogenic bacteria such as Methanosarcina species are able to grow on analogous Cl feedstocks including methanol and methylamine by using variations of this same pathway for carbon assimilation. The key enzyme in these cases is carbon monoxide dehydrogenase, a complex nickel enzyme so named for its ability to catalyze the reduction of CO2 to CO. However, the enzyme has been implicated in a number of cases as the catalyst for the carbon-carbon bond formation from suitable methyl donors and enzymatically generated CO from CO2 to give acetyl units.In the acetogenic Clostridium thermoaceticum, one molecule of CO2 is reduced to formate and formate is reduced to a methyl group by using tetrahydrofolate as a carrier; enzymatic transfer to a corrinoid iron/sulfur protein yields the methylated organocobalt species (19). Nickel-containing CO dehydrogenase (more appropriately called acetyl-CoA synthase) then acts as the site of carbon-carbon bond coupling by accepting the methyl group and catalyzing its carbonylation in the presence of CoA to give acetyl-CoA as product. The enzyme from C. thermoaceticum is the most well characterized acetogenic enzyme containing 2 Ni, 11 Fe, and 1 Zn atoms per active site; there is some discussion about the subunit composition of the functional complex as isolated: c433 [

The cytoplasmic domains of two human transmembrane protein tyrosine phosphatases (PTPases), LAR and CD45, have been expressed in Escherichia coli, purified to near-homogeneity, and compared for catalytic efficiency toward several phosphotyrosine-containing peptide substrates. A 615-residue LAR fragment (LAR-D1D2) containing both tandemly repeated PTPase domains shows almost identical specific activity and high catalytic efficiency as the 40-kDa single-domain LAR-D1 fragment, consistent with a single functional active site in the 70-kDa LAR-D1D2 enzyme. A 90-kDa fragment of the human leukocyte CD45 PTPase, containing two similar tandemly repeated PTPase domains, shows parallel specificity to LAR-D1 and LAR-D1D2 with a high kcat/Km value for a phosphotyrosyl undecapeptide. Sufficient purified LAR-D1 and LAR-D1D2 PTPases were available to demonstrate enzymatic exchange of 18O from 18O4 inorganic phosphate into H2(16)O at rates of approximately 1 x 10(-2) s-1. The oxygen-18 exchange probably proceeds via a phosphoenzyme intermediate. Brief incubation of all three PTPase fragments with a [32P]phosphotyrosyl peptide substrate prior to quench with SDS sample buffer and gel electrophoresis led to autoradiographic detection of 32P-labeled enzymes. Pulse/chase studies on the LAR 32P-enzyme showed turnover of the labeled phosphoryl group.

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