Bruce A. Averill

Hydrolysis of phosphate monoesters: a biological problem with multiple chemical solutions.

Formation of phosphate esters by kinases has long been recognized as an important process in biochemistry, but the reverse reaction, hydrolysis of phosphate esters by phosphatases, has attracted less attention. Recent work suggests that phosphatases are as important as kinases in regulatory processes, and that they constitute a diverse group of enzymes that utilize a variety of chemical means to accelerate phosphate ester hydrolysis.

Evidence that the type 2 copper centers are the site of nitrite reduction by Achromobacter cycloclastes nitrite reductase

Methods have been developed for selective depletion and reconstitution of the Type 2 Cu (non-blue) sites in the nitrite reductase from A. cycloclastes, resulting in preparations ranging from 0.5 to 2.6 Type Cu per trimer; the Type 1 Cu content is invariant at 3.0 per trimer. The activity of the enzyme is directly proportional to the Type 2 content as measured by direct metal determination or by analysis of the EPR spectra. These results indicate that an earlier report that the A. cycloclastes enzyme contains only Type 1 Cu sites is incorrect, and that the Type 2 Cu centers constitute the site at which NO2- is reduced. Furthermore, they suggest that other Cu nitrite reductases that are reported to contain only Type 1 Cu sites and exhibit relatively low activity may actually be largely Type 2 Cu-depleted forms of the enzymes.

Evidence for an NO-rebound, mechanism for production of N2O from nitrite by the copper-containing nitrite reductase from Achromobacter cycloclastes

Reduction of NO− 2 by the Cu‐containing nitrite reductase from Achromobacter cycloclastes produces NO as the primary product initially, but as NO accumulates, NO production levels‐off and N2O production becomes significant. Reaction of the enzyme with NO− 2 in the presence of NO increases the amount of N2O product significantly, while trapping the NO product as nitrosylhemoglobin or rapid, removal of NO by sparging results in no detectable N2O production. Reaction of the enzyme with 15NO− 2 in the presence of 14NO results in rapid formation of the mixed isotope product (14N, 15N)O in ca. 45% yield. In contrast, the presence or absence of NO has no effect on N2O production by a prototypical heme cd 1‐containing nitrite reductase. These results are consistent with formation of a labile Cu+−NO+ species in the copper enzyme, which normally decomposes to NO. Production of N2O requires that the released NO must rebind to the enzyme to combine with a second NO− 2 or a species derived therefrom.

Raman characterization of human leukocyte myeloperoxidase and bovine spleen green haemoprotein. Insight into chromophore structure and evidence that the chromophores of myeloperoxidase are equivalent

Soret excitation resonance Raman spectroscopy has been used to characterize dimeric human leukocyte myeloperoxidase (donor:hydrogen peroxide oxidoreductase, EC 1.11.1.7) and monomeric bovine spleen green haemoprotein. The spectra of the two proteins, under the same conditions of iron valence and ligation, are essentially identical. Owing to strong symmetry reduction effects, the spectra are more complex than usually observed for haemoproteins. It is possible, however, to assign the high-frequency vibrations and, from these assignments, to determine structural features of the iron chromophores. In the resting protein, the iron adopts a six-coordinate high-spin configuration in both proteins; cyanide addition produces six-coordinate low-spin species, and in the ferrous enzymes the iron appears to be five-coordinate and high-spin. The proteins are stable to laser excitation and do not photoreduce under illumination. No evidence is found for unusual peripheral substituents, such as formyl or protonated Schiffs base group, in conjugation with the main chromophore in the native protein. The vibrational data are consistent with an iron chlorin chromophore, although other electronic effects, in addition to those produced by porphyrin ring reduction, are necessary to account for the optical properties of the proteins. The similarity in Raman spectra for myeloperoxidase and green haemoprotein indicates that the two iron sites in myeloperoxidase are equivalent.

Sequence homology in the metalloproteins; purple acid phosphatase from beef spleen and uteroferrin from porcine uterus

The primary structures of purple acid phosphatase and uteroferrin, two iron-binding glycoproteins isolated from beef spleen and porcine uterine fluids, respectively, have been examined by a combination of tandem mass spectrometry and classical Edman sequencing methods. Reported here are amino acid sequence data covering more than 90% of the primary structures for these two proteins. The sequence data reveal an unexpectedly high degree of homology, greater than 90%, for these two proteins.

Sequence homology between purple acid phosphatases and phosphoprotein phosphatases Are phosphoprotein phosphatases metalloproteins containing oxide-bridged dinuclear metal centers?

The amino acid sequences of mammalian purple acid phosphatases and phosphoprotein phosphatases are shown to possess regions of significant homology. The conserved residues contain a high percentage of possible metal‐binding residues. The phosphoprotein phosphatases 1, 2A and 2B are proposed to be iron‐zinc metalloenzymes with active sites isostructural (or nearly so) with those of the purple phosphatases.

The highly exposed loop region in mammalian purple acid phosphatase controls the catalytic activity.

Recombinant human purple acid phosphatase (recHPAP) provides a convenient experimental system for assessing the relationship between molecular structure and enzymatic activity in mammalian purple acid phosphatases (PAPs). recHPAP is a monomeric protein with properties similar to those of uteroferrin (Uf) and other PAPs isolated as single polypeptide chains, but its properties differ significantly from those of bovine spleen PAP (BSPAP) and other PAPs isolated as proteolytically “clipped” forms. Incubation of recHPAP with trypsin results in proteolytic cleavage in an exposed region near the active site. The product is a tightly associated two‐subunit protein whose collective spectroscopic and kinetics properties resemble those of BSPAP. These results demonstrate that the differences in spectroscopic and kinetics properties previously reported for mammalian PAPs are the result of proteolytic cleavage. Mass spectrometry shows that a three‐residue segment, D‐V‐K, within the loop region is excised by trypsin. This finding suggests that important interactions between residues in the excised loop and one or more of the groups that participate in catalysis are lost or altered upon proteolytic cleavage. Analysis of available structural data indicates that the most important such interaction is that between Asp 146 in the exposed loop and active‐site residues Asn 91 and His 92. Loss of this interaction should result in both an increase in the Lewis acidity of the FeII ion and an increase in the nucleophilicity of the FeIII‐bound hydroxide ion. Proteolytic cleavage thus constitutes a potential physiological mechanism for regulating the activity of PAP in vivo.

Isolation of a high specific activity pink, monomeric nitrous oxide reductase from Achromobacter cycloclastes

Nitrous oxide reductase from Achromobacter cycloclastes has been purified to homogeneity under aerobic conditions via DEAE-cellulose, phenyl-Sepharose, hydroxyapatite, and Sephacryl S-200 chromatography. It consists of a single polypeptide of MW 72 kDa, and contains 3.8 +/- 0.1 copper atoms per molecule. The enzyme is pink as isolated, yet exhibits a specific activity (86 U/mg) that is ca. 40 times greater than that observed for other N2O reductases under similar conditions. Double integration of the anomalous EPR spectrum at 77K showed the presence of 2.0 +/- 0.1 spins per molecule, implying the presence of EPR-silent copper atoms and/or spin-coupled mixed-valent centers.

Substrate positioning by His92 is important in catalysis by purple acid phosphatase

Proteolysis of single polypeptide mammalian purple acid phosphatases (PAPs) results in the loss of an interaction between the loop residue Asp146 and the active site residues Asn91 and/or His92. While Asn91 is a ligand to the divalent metal of the mixed‐valent di‐iron center, the role of His92 in the catalytic mechanism is unknown. Site‐directed mutagenesis of His92 was performed to examine the role of this residue in single polypeptide PAP. Conversion of His92 into Ala, which eliminates polar interactions of this residue with the active site, resulted in a 10‐fold decrease in catalytic activity at the optimal pH. Conversely, conversion of this residue into Asn, which cannot function as either a proton donor or acceptor, but can provide hydrogen–bonding interactions, resulted in a three‐fold increase in activity at the optimal pH. Both mutant enzymes had more acidic pH optima, with pKes,1 values consistent with the involvement of an iron(III) hydroxide unit or a hydroxide in the second coordination sphere in catalysis. These results, together with EPR data, support a role of His92 in positioning either the nucleophile or the substrate, rather than directly in acid or base catalysis. The existence of an extensive hydrogen‐bonding network that could fine‐tune the position of His92 is consistent with this proposal.

A spectrophotometric assay for dissimilatory nitrite reductases

A spectrophotometric assay for dissimilatory nitrite reductases has been developed utilizing mammalian cytochrome c (equine heart) as reductant and spectrophotometric agent. The copper-containing nitrite reductase from Achromobacter cycloclastes has been shown to have apparent Kms for reduced cytochrome c and nitrite of 86 +/- 5 and 5.63 +/- 0.03 microM, respectively. The heme cd-containing enzyme from Pseudomonas stutzeri was shown to have apparent Kms for reduced cytochrome c and nitrite of 260 +/- 60 and 1.8 +/- 0.8 microM, respectively. This assay represents a simple, general method for consistently evaluating the activity of the copper- and heme cd-containing nitrite reductases that are capable of utilizing readily available mammalian cytochrome c as electron donor and should be useful for mechanistic studies of these enzymes.