Subunits catalase

Fig. 8. Comparison of the subunit structures of a small subunit catalase (BLC) (A) and a large subunit catalase (HPII) (B). Segments including the N-terminal domain, the beta barrel core, the wrapping domain, the alpha helical domain, and the C-terminal domain (HPII) are indicated and are described in Section V,A.

The heme of catalases is deeply buried within the core of the catalase subunit. Protoheme IX or heme b is found in all small-subunit catalases so far characterized. The two large-subunit enzymes HPII and PVC have been characterized biochemically, spectrally, and structurally 91) as containing heme d in which ring HI is oxidized to a cis-hydroxyspirolactone. Heme b is initially bound to both enzymes during assembly, and it is subsequently oxidized by the catalase itself during the early rounds of catalysis 92). 

Fig. 13. Active site residues in a small-subunit catalase BLC (A) and a large-subunit catalase HPIl (B). The active site residues are labeled, and hydrogen bonds are shown between the serine (113 in BLC and 167 in HPll) and the essential histidine (74 in BLC and 128 in HPll). A single water is shown hydrogen bonded to the histidine. The equivalent water in BLC is located by analogy to the position of the water in HPll. The unusual covalent bond between the N of His392 and the C of Tyr415 in HPll is evident on the proximal side of the heme in B. The flipped orientations of the hemes are evident in a comparison of the two structures, as is the eis-hydroxyspirolactone structure of heme d in B.

Fig. 16. A cartoon showing the putative channels that provide access to the active site of a large-subunit catalase in A and a small-subunit catalase in B. The main or perpendicular channel is labeled P and the minor or lateral channel, which is bifurcated, is labeled L. A potential channel leading to the proximal side of the heme is shown with a dashed line.

The modification of heme b to heme d 91, 92) observed in the large-subunit enzymes HPII and PVC has already been discussed in detail. Whether all large-subunit enzymes will be found to contain such a modified heme remains to be seen, and the fact that HPII variants containing heme b retain activity suggests that naturally occurring large subunit catalases with heme b may be found. 

Each subunit of a heme catalase binds a single molecule of heme and some mammalian catalases also possess a second cofactor, NADPH. The binding of NADPH in catalases was at first totally unexpected, but has since been a frequent feature of small-subunit catalases from both prokaryotic and eukaryotic organisms. However, the actual biochemical function of NADPH in these catalases is still not fully understood. One possible role is protection of the enzyme against inactivation by its own substrate, especially under conditions of low-peroxide concentrations. The NADPH binding pocket is located on the molecular surface, just above an entrance chaimel with the nicotinamide active carbon situated approximately 20 A from the closest heme atom (Figure ll(a)). ... 

The diversity among catalases, evident in the variety of subunit sizes, the number of quaternary structures, the different heme prosthetic groups, and the variety of sequence groups, enables them to be organized in four main groups the classic monofunctional enzymes (type A), the catalase-peroxidases (type B), the nonheme catalases (type C), and miscellaneous proteins with minor catalatic activities (type D). 

Within this common core structure, modifications have been identified that provide catalases with further unique properties. The large-subunit enzymes have extensions at both the amino and carboxyl ends, the latter having a flavodoxinlike structure, a unique His-Tyr bond, a protected cysteine, and a modified heme. NADPH binding and an oxidized methionine are found in small subunit enzymes. Identification and assignment of roles to channels providing access to and egress from the deeply buried heme have recently become the focus of study. Analysis of the structure of catalase HPII of E. coli has been facilitated by the construction of more than 75 mutants (Table I). 

The tertiary structure of small subunit enzymes can be subdivided into four distinct regions, and the C-terminal or flavodoxin domain of the large subunit enzymes becomes a fifth region. These are indicated in Fig. 8 for clarity. The first region is the amino terminal arm (Fig. 8), which extends 50 or more residues from the amino terminus almost to the essential histidine residue (to residue 53 in PMC, 60 in PVC, 73 in BLC, and 127 in HPII). There is very little structural similarity in the N-terminal region and, in the case of HPII, the structure of the terminal 27 residues is not even defined and they do not appear in the crystal structure. Within the N-terminal arm is a 20-residue helix, helix a2 in HPII, which is the first secondary structure element common to all catalases. The presence of helix al varies among catalases, and there is no sequence or location equivalence even when it is present. 

Fig. 9. Comparison of the dimensions of small-subunit (BLC) and large-subunit (HPll) catalases. BLC is shown in panels A and B, in the R-P and R-Q orientations, respectively HPll is shown in C and D, also in the R P and R-Q orientations, respectively.

Bergdoll et al. 87) have proposed that some proteins, including catalase, exhibit arm exchange or an interaction of one subunit with an adjacent subimit to stabilize quaternary structure, and that this... 

Fig. 12. A hypothetical folding and assembly pathway for catalases. In A secondary and tertiary folding first occurs in the individual subunits to form the 3-barrel (p), wrapping domain (W), a-helical segment (a), and fiavodoxin domain (F, only in HPII). In proceeding to B, heme is bound to each of the subunits, and this may serve as a catalyst for the rapid association of the i -related subunits to form the structure in C. In proceeding to D, Q-related subunits associate, resulting in the N-terminal arms being overlapped as the C-terminal portions fold back on themselves to form the fully folded structure shown in E. Only two subunits are shown in the progression from C to E, but a simultaneous folding must be occurring in the associated dimer. The fully folded tetramer is shown in two orientations.

Have we exhausted catalases as a source of information about protein structure and the catalatic mechanisms The answer is clearly no. With each structure reported comes new information, often including structural modifications seemingly unique to catalases and with roles that remain to be explained. Despite a deeply buried active site, catalases exhibit one of the fastest turnover rates determined. This presents the as yet unanswered question of how substrate can access the active site while products are simultaneously exhausted with a potential turnover rate of up to 10 per second. The complex folding pathway that produces the intricate interwoven arrangement of subunits also remains to be fully clarified. 

Crystal structures of manganese catalases (in the (111)2 oxidation state) from Lactobacillus plantarum,its azide-inhibited complex, " and from Thermus thermophilus have been determined. There are differences between the structures that may reflect distinct biological functions for the two enzymes, the L. plantarum enzyme functions only as a catalase, while the T. thermo-philus enzyme may function as a catalase/peroxidase. The active sites are conserved in the two enzymes and are shown schematically in Figure 32. Each subunit contains an Mu2 active site,... 

CGD) Defect due to mutation in neutrophil enzyme NADPH oxidase gene coding for any one of the four subunits of the enzyme Patients subject to recurrent infections by catalase-positive organisms and to granuloma formation X-Unked mutation more severe... 

Identical subunits Glyceraldehyde-3-phosphate dehydrogenase, catalase, alcohol dehydrogenase, hexokinase Mostly intracellular enzymes rarely have disulfide bonds... 

As for the peroxidases, Compound I and water are formed in the first step from one equivalent of hydrogen peroxide and the resting state of the catalase. The back-reaction, however, does not proceed via Compound II but rather via a two-electron-two-proton transfer cascade, in which both hydrogen atoms of a second molecule of hydrogen peroxide are transferred to the ferryl subunit of the porphyrin cofactor. Due to the similarity of catalases and peroxidases, it is not too surprising that this reaction is also catalyzed by most peroxidases. Alternatively, catalases and some peroxidases react with alkyl hydroperoxides via the respective alkanol to an aldehyde or ketone (Scheme 2.17). A requirement for this reaction is an easily accessible active site for the hydroperoxide, so that only those peroxidases with open access such as CPO or CiP are able to promote this reaction. 

Let us now consider the questions of the biological oxidation of various substrates with hydrogen peroxide in the presence of catalases and peroxidases. As Pratt notes [82], this group of enzymes is unique in that it is the only one in which intermediates are detected, and all stages of catalytic process are determined, identified and studied. Of special value is the characteristic that catalase consists of four subunits, whereas peroxidase possesses only one subunit. Using special technique, it is also shown that every iron atom (heme) binds one H202 molecule [83 ]. 

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