David S. Auld

Fluorescence determination of carboxypeptidase A activity based on electronic energy transfer

Abstract Resonance energy transfer between suitable donor and acceptor groups can occur over distances of 20 A or more, provided the donor fluoresces efficiently in a wavelength range over which the acceptor absorbs strongly. Hence, a chromophoric quenching group can be introduced into the substrate at a distance remote from the susceptible bond. The peptide region in the neighborhood of the bond to be broken can then be designed in accord with the specificity requirements of the particular enzyme. These specifications have been met through the synthesis of a series of Dns-(Gly) n -Trp substrates for carboxypeptidase A. The present assay is based on energy transfer and is applicable to other substrates of carboxypeptidase A and to other hydrolytic enzymes.

Yeast RNA polymerase I: A eukaryotic zinc metalloenzyme

Abstract Microwave excitation spectrometry and metal binding inhibition studies show that zinc is a catlytically essential component of the highly purified RNA polymerase I from yeast, the first eukaryotic RNA polymerase I available in quantities sufficient for such studies. It contains 2.4 g-atom of zinc based on a molecular weight of 6.5 × 10 5 (8). Copper, iron, manganese and magnesium are absent, i.e., below the limits of detection, 10 −13 to 10 −14 g-atoms. A number of derivatives of 1,10-phenanthroline reversibly inhibit the polymerase catalyzed reaction, apparently by forming a ternary polymerase·Zn·OP complex while the nonchelating isomer, 1,7-phenanthroline, is ineffective.

Zinc reverse transcriptases from mammalian RNA type C viruses.

1,10-phenanthroline (OP) reversibly inhibits the RNA dependent DNA polymerases reverse transcriptases from murine, simian, feline and RD-114 RNA tumor viruses. The log of the OP inhibition constants, pK I , vary from 4.7 to 4.9 and the number of moles of inhibitor bound to the metal, ii, vary from 1.3 to 2.2. These values are very similar to those previously reported for known zinc enzymes and imply that the mammalian reverse transcriptases are also zinc enzymes. Combined use of microwave-induced emission spectrometry and micro gel exclusion chromatography demonstrates the presence of stoichiometric amounts of zinc in the enzymes from murine leukemia and woolly monkey type C-viruses. These results parallel those previously reported for the enzyme from avian myeloblastosis virus.

The effect of modifiers on the hydrolysis of esters and peptides by carboxypeptidase A.

Abstract In the course of kinetic investigations of carboxypeptidase A we have found that N-substituted products of hydrolysis of certain synthetic peptide and ester substrates activate peptidase but inhibit esterase activity. Similar effects have been observed with a number of compounds, referred to collectively as modifiers, which have properties analogous to those of such N-terminal blocking groups (2,10–12). The present communication extends our previous reports concerning these phenomena and relates them to the kinetic data currently available on carboxypeptidase catalysis.

Methionyl-tRNA synthetase of Escherichia coli A zinc metalloprotein

The native dimeric form of methionyl-tRNA synthetase of Escherichia coli contains two zinc atoms per dimer, one per subunit. The bound zinc is retained upon trypsin modification which yields a monomer with one zinc atom. The enzymatic activity of both the dimeric forms is reversibly inhibited by 1,10-phenanthroline but not by its non-chelating analogues. In addition, the native enzyme binds two Mn2+ per dimer with a binding constant of approx. 70 micron but no binding is observed with the trypsin-modified monomer.

Reverse transcriptase from avian myeloblastosis virus: A zinc metalloenzyme

Abstract Previous postulates of a relationship between a zinc enzyme and the leukemic process (1,2) have led to the identification of the RNA dependent DNA polymerase -- reverse transcriptase -- of avian myeloblastosis virus as a zinc metalloenzyme. Microwave induced emission spectrometry provides a microanalytical system capable of measuring precisely 10 −11 to 10 −14 g atoms of metal in microgram amounts of enzyme, orders of magnitude more sensitive than other, conventional methods. The chromatographic fraction with highest enzymatic activity contains 1.5 × 10 −11 g atoms of zinc per 1.4 μg of protein, corresponding to 1.7 to 1.9 g atoms of zinc per mole of enzyme for a molecular weight previously determined either as 1.6 or 1.8 × 10 5 . The Zn/activity ratio is constant in the active fractions. Copper, iron and manganese are absent, i.e., at or below their limits of detection, 10 −13 to 10 −14 g atoms. Agents known to chelate zinc inhibit the enzyme while their nonchelating isomers do not. The data underline the participation of zinc in nucleic acid metabolism and bear importantly upon the lesions which accompany leukemia and zinc deficiency.

Zinc catalysis in metalloproteases

Analyses of zinc protease sites using X-ray diffraction, NMR and X-ray absorption fine structure in combination with functional studies of these enzymes are beginning to reveal the manner in which the protein modulates the properties of the zinc to achieve specificity and catalytic efficiency. The chemistry of zinc: 1) permits a variety of types and numbers of ligands and coordination geometries, 2) promotes ionization of water at neutral pH values and 3) is inert to oxido-reductants present in the medium. The type of ligands and the protein scaffolding of the zinc binding site determine the binding strength of zinc and its catalytic properties. These properties in turn are critical to the role of zinc in a wide variety of metalloexo- and endo-protease-catalyzed processes.

Low temperature stopped-flow spectrometry.

Abstract A stopped-flow instrument operating over the temperature range from +55 to as low as −55°C features a Teflon stopped-flow module with a quartz flow-through cell, which is placed in a heavy, double-walled, air-insulated, Plexiglas environmental chamber. Nitrogen gas is the temperature control fluid. The combination of the Teflon components, quad x-rings, and fluorinated lubricant results in a flow system which is inert, metal free, and has proven to be leak free at temperatures as low as −55°C. Quartz fiber optics connect the observation cell with the external light supply and detection systems which can monitor fluorescence or absorbance of the sample solution. The performance of the instrument at subzero temperatures has been evaluated by measuring the rate of the sodium hydroxide catalyzed hydrolysis of the ester, o -nitrophenylchloroacetate, at 430 nm. At −20°C, in 70% methanol-water the first-order rate constant increases linearly from 2.7 to 81 s −1 over the hydroxide concentration range 5–150 m m . Even at −55°C the flow process is artifact free and complete in a few milliseconds. A linear Arrenhius plot is obtained for ester hydrolysis by 5 m m hydroxide over the temperature range from +20 to −55°C.

Protease susceptibility of zinc - and APO-carboxypeptidase A

Proteases in preparations of carboxypeptidase A progressively inactivate solutions of the apoenzyme but not the metal-containing enzyme. Free amino acids generated by proteolysis interfere with spectral studies after reconstituting the apoenzyme with cobalt. Purification by affinity chromatography eliminates this effect. Affinity-purified apoenzyme is susceptible to digestion with chymotrypsin but the metalloenzyme is not.

Essential arginyl residues in reverse transcriptase

Abstract The RNA dependent DNA polymerase -- reverse transcriptases -- from avian, feline and simian type C RNA viruses are rapidly inactivated by butanedione in borate buffer, a reagent which is highly selective for the modification of arginyl residues. The pronounced enhancement by borate on the inactivation of reverse transcriptase implicates essential arginyl residues, and substrate protection experiments with the polymerase from avian myeloblastosis virus indicate that these residues function in binding the RNA template to the active site.