Flexibility in substrate recognition by thimet oligopeptidase as revealed by denaturation studies.

Thimet oligopeptidase (TOP) is a soluble metalloendopeptidase belonging to a family of enzymes including neurolysin and neprilysin that utilize the HEXXH metal-binding motif. TOP is widely distributed among cell types and is able to cleave a number of structurally unrelated peptides. A recent focus of interest has been on structure-function relationships in substrate selectivity by TOP. The enzyme's structural fold comprises two domains that are linked at the bottom of a deep substrate-binding cleft via several flexible loop structures. In the present study, fluorescence spectroscopy has been used to probe structural changes in TOP induced by the chemical denaturant urea. Fluorescence emission, anisotropy and collisional quenching data support a two-step unfolding process for the enzyme in which complete loss of the tertiary structure occurs in the second step. Complete loss of activity and loss of catalytic Zn(II) from the active site, monitored by absorption changes of the metal chelator 4-(2-pyridylazo)-resorcinol, are also connected with the second step. In contrast, the first unfolding event, which is linked to changes in the non-catalytic domain, leads to a sharp increase in kcat towards a 9-residue substrate and a sharp decrease in kcat for a 5-residue substrate. Thus a conformational change in TOP has been directly correlated with a change in substrate selectivity. These results provide insight into how the enzyme can process the range of structurally unrelated peptides necessary for its many physiological roles.

pH dependence studies provide insight into the structure and mechanism of thimet oligopeptidase (EC 3.4.24.15).

Thimet oligopeptidase (EC 3.4.24.15; TOP) is a Zn(II) endopeptidase implicated in physiological regulation of processes involving neuropeptides. The present study clarifies the active site structure and mechanism of catalysis of TOP. The enzyme exhibited a bell-shaped pH dependence of activity having an acidic limb due to a protonation event with a pK(a) of 5.7 and a basic limb with pK(a) of 8.8. The acidic limb can be attributed to protonation of a residue affecting k(cat) while the alkaline limb may be due to conformational change. Mutation of Tyr612 to Phe resulted in more than 400-fold decrease in activity. This result, supported by modeling studies, implicates Tyr612 in transition state stabilization analogous to the role of His231 of thermolysin.

Thermodynamic properties of peptide folding.

Inter- and intramolecular forces and thermodynamics are concepts usually presented early within introductory biochemistry and have relevance to many topics throughout the remainder of the course. For this reason, it is important to have a good foundation in these concepts so they can be applied to many systems. To this end, a group problem has been developed that allows students to analyze thermodynamic data and, using their knowledge of inter- and intramolecular forces, gain an understanding of the elements involved in the thermodynamic stability and folding of a peptide.

Hydrogen bond residue positioning in the 599-611 loop of thimet oligopeptidase is required for substrate selection.

Thimet oligopeptidase (EC 3.4.24.15) is a zinc(II) endopeptidase implicated in the processing of numerous physiological peptides. Although its role in selecting and processing peptides is not fully understood, it is believed that flexible loop regions lining the substrate-binding site allow the enzyme to conform to substrates of varying structure. This study describes mutant forms of thimet oligopeptidase in which Gly or Tyr residues in the 599-611 loop region were replaced, individually and in combination, to elucidate the mechanism of substrate selection by this enzyme. Decreases in k(cat) observed on mutation of Tyr605 and Tyr612 demonstrate that these residues contribute to the efficient cleavage of most substrates. Modeling studies showing that a hinge-bend movement brings both Tyr612 and Tyr605 within hydrogen bond distance of the cleaved peptide bond supports this role. Thus, molecular modeling studies support a key role in transition state stabilization of this enzyme by Tyr605. Interestingly, kinetic parameters show that a bradykinin derivative is processed distinctly from the other substrates tested, suggesting that an alternative catalytic mechanism may be employed for this particular substrate. The data demonstrate that neither Tyr605 nor Tyr612 is necessary for the hydrolysis of this substrate. Relative to other substrates, the bradykinin derivative is also unaffected by Gly mutations in the loop. This distinction suggests that the role of Gly residues in the loop is to properly orientate these Tyr residues in order to accommodate varying substrate structures. This also opens up the possibility that certain substrates may be cleaved by an open form of the enzyme.

The role of copper and protons in heme-copper oxidases: kinetic study of an engineered heme-copper center in myoglobin.

To probe the role of copper and protons in heme-copper oxidase (HCO), we have performed kinetic studies on an engineered heme-copper center in sperm whale myoglobin (Leu-29 --> HisPhe-43 --> His, called Cu(B)Mb) that closely mimics the heme-copper center in HCO. In the absence of metal ions, the engineered Cu(B) center in Cu(B)Mb decreases the O(2) binding affinity of the heme. However, addition of Ag(I), a redox-inactive mimic of Cu(I), increases the O(2)-binding affinity. More importantly, copper ion in the Cu(B) center is essential for O(2) reduction, as no O(2) reduction can be observed in copper-free, Zn(II), or Ag(I) derivatives of Cu(B)Mb. Instead of producing a ferryl-heme as in HCO, the Cu(B)Mb generates verdoheme because the engineered Cu(B)Mb may lack a hydrogen bonding network that delivers protons to promote the heterolytic OO cleavage necessary for the formation of ferryl-heme. Reaction of oxidized Cu(B)Mb with H(2)O(2), a species equivalent in oxidation state to 2e(-), reduced O(2) but, possessing the extra protons, resulted in ferryl-heme formation, as in HCO. The results showed that the Cu(B) center plays a critical role in O(2) binding and reduction, and that proton delivery during the O(2) reduction is important to avoid heme degradation and to promote the HCO reaction.

Neutral thiol as a proximal ligand to ferrous heme iron: implications for heme proteins that lose cysteine thiolate ligation on reduction.

Cysteine plays a key role as a metal ligand in metalloproteins. In all well-recognized cases, however, it is the anionic cysteinate that coordinates. Several cysteinate-ligated heme proteins are known, but some fail to retain thiolate ligation in the ferrous state, possibly following protonation to form neutral cysteine. Ligation by cysteine thiol in ferrous heme proteins has not been documented. To establish spectroscopic signatures for such systems, we have prepared five-coordinate adducts of the ferrous myoglobin H94G cavity mutant with neutral thiol and thioether sulfur donors as well as six-coordinate derivatives such as with CO and, when possible, with NO and O(2). A thiol-ligated oxyferrous complex is reported, to our knowledge for the first time. Further, a bis-thioether ferrous H93G model for bis-methionine ligation, as found in Pseudomonas aeruginosa bacterioferritin heme protein, is described. Magnetic CD spectroscopy has been used due to its established ability in axial ligand identification. The magnetic CD spectra of the H93G complexes have been compared with those of ferrous H175CD235L cytochrome c peroxidase to show that its proximal ligand is neutral cysteine. We had previously reported this cytochrome c peroxidase mutant to be cysteinate-ligated in the ferric state, but the ferrous ligand was undetermined. The spectral properties of ferrous liver microsomal cytochrome P420 (inactive P450) are also consistent with thiol ligation. This study establishes that neutral cysteine can serve as a ligand in ferrous heme iron proteins, and that ferric cysteinate-ligated heme proteins that fail to retain such ligation on reduction may simply be ligated by neutral cysteine.