Polymorphonuclear neutrophils generate both nitric oxide and superoxide and these molecules can combine to form peroxynitrite. Neutrophils also contain myeloperoxidase which reacts with peroxynitrous acid (HOONO). On mixing myeloperoxidase with HOONO compound I1 was formed. Compound I could not be detected as an intermediate. The apparent second-order rate constant of formation of compound I1 was strongly pH-dependent (2.5 X lo5 M-' . s-' at pH 8.9 and 6.2 X 106 M-' . s-' at pH 7.2). The pK, of this effect is 6.9 and it was concluded that the enzyme reacts with the protonated form of the peroxide, that is peroxynitrous acid, with a pH-independent second-order rate constant of 2.0X lo7 M-' . s-' at 12°C. The interaction of HOONO with lactoperoxidase was studied for comparison. As was observed for myeloperoxidase, compound I could not be detected as an intermediate. The apparent second-order rate constant of compound I1 formation is pH-dependent and is 3.3 X lo5 M-' . s-' at pH 7.4 and 8.4 X lo4 M-' . s-' at pH 9.0. In contrast, horseradish peroxidase reacts with HOONO to form compound I, which is subsequently followed by the formation of compound 11. The second-order rate constant for the formation of compound I is 3.2 X lo6 M-' . s-' and is pH-dependent, the pK, for this effect is 6.8. Catalase (up to 3 pM) does not affect the rate of decomposition of peroxynitrite and no compound I formation is observed. Since nitrite may be present in the peroxynitrite preparation and to discriminate between the reaction of the enzyme with nitrite or peroxynitrite, the effect of nitrite on myeloperoxidase was studied. The dissociation constant for the myeloperoxidase-nitrite complex is pH-dependent and has values of 580 pM at pH 6.0 and 55 mM at pH 8.5.Since the recent discovery that the biological activity of the endothelium-derived relaxing factor is accounted for by nitric oxide (NO) [l-31 the interest in NO has greatly increased 141. To date nitric oxide has been shown to be involved as a bioregulatory molecule in many different biological functions such as vascular smooth muscle relaxation, platelet deaggregation 151 and neural communication [6, 71. Nitric oxide is also synthesized by murine macrophages and neutrophils [8, 91 in a fashion similar to its formation in vascular endothelial cells. The molecule is synthesized from one of the two chemically equivalent terminal guanidino nitrogen atoms from L-arginine by the enzyme NO synthase, classified into two groups, constitutive and inducible, of which the constitutive enzymes are Ca2+/calmodulin-dependent [lo-131. ln contrast, the inducible NO synthase from macrophages [ 14, 151 and that from polymorphonuclear neutrophils 11 61 are calmodulin-independent, though both the inducible and the constitutive type of NO synthases are dependent on NADPH. Very recently the first NO synthase has been purified and spectroscopically characterized and has

The binding of penicillin to penicillin acylase was studied by X-ray crystallography. The structure of the enzyme-substrate complex was determined after soaking crystals of an inactive betaN241A penicillin acylase mutant with penicillin G. Binding of the substrate induces a conformational change, in which the side chains of alphaF146 and alphaR145 move away from the active site, which allows the enzyme to accommodate penicillin G. In the resulting structure, the beta-lactam binding site is formed by the side chains of alphaF146 and betaF71, which have van der Waals interactions with the thiazolidine ring of penicillin G and the side chain of alphaR145 that is connected to the carboxylate group of the ligand by means of hydrogen bonding via two water molecules. The backbone oxygen of betaQ23 forms a hydrogen bond with the carbonyl oxygen of the phenylacetic acid moiety through a bridging water molecule. Kinetic studies revealed that the site-directed mutants alphaF146Y, alphaF146A and alphaF146L all show significant changes in their interaction with the beta-lactam substrates as compared with the wild type. The alphaF146Y mutant had the same affinity for 6-aminopenicillanic acid as the wild-type enzyme, but was not able to synthesize penicillin G from phenylacetamide and 6-aminopenicillanic acid. The alphaF146L and alphaF146A enzymes had a 3-5-fold decreased affinity for 6-aminopenicillanic acid, but synthesized penicillin G more efficiently than the wild type. The combined results of the structural and kinetic studies show the importance of alphaF146 in the beta-lactam binding site and provide leads for engineering mutants with improved synthetic properties.

One of the cell types first encountered by human immunodeficiency virus type 1 (HIV-1) following sexual transmission are dendritic cells (DC). DC capture HIV-1 through C-type lectin receptors, of which the best studied example is DC-SIGN, which mediates HIV-1 internalization. DC can keep the virus infectious for several days and are able to transmit HIV-1 to CD4؉ T cells. We tested proteins from milk and serum for their ability to block DC-mediated HIV-1 transmission, of which bovine lactoferrin (bLF) is the most potent inhibitor. bLF binds strongly to DC-SIGN, thus preventing virus capture and subsequent transmission. Interestingly, bLF is a much more efficient inhibitor of transmission than human lactoferrin. Since bLF is nontoxic and easy to purify in large quantities, it is an interesting candidate microbicide against HIV-1. Another advantage of bLF is its ability to block HIV-1 replication in T cells. DC-mediated capture of a bLF-resistant HIV-1 variant that was selected during long-term culturing in T cells could still be blocked by bLF. This underscores the usefulness of bLF as a microbicide drug to prevent HIV-1 transmission.

The observation that the bitterest peptides from casein hydrolysates contain several proline residues led us to hypothesize that a proline-specific protease would be instrumental in debittering such peptides. To identify the desired proline-specific activity, a microbiological screening was carried out in which the chromogenic peptide benzyloxycarbonyl-glycine-proline-p-nitroanilide (Z-Gly-Pro-pNA) was used as the substrate. An Aspergillus niger (A. niger) strain was identified that produces an extracellular proline-specific protease with an acidic pH optimum. On the basis of sequence similarities, we conclude that the A. niger-derived enzyme probably belongs to the S28 family of clan SC of serine proteases rather than the S9 family to which prolyl oligopeptidases belong. Incubating the overexpressed and purified enzyme with bitter casein hydrolysates showed a major debittering effect. Reversed phase HPLC analysis revealed that this debittering effect is accompanied by a significant reduction of the number of hydrophobic peptides present.

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