Eukaryotic enzymes

Several catalases, including the type B catalase-peroxidases, seem to show true substrate saturation at much lower levels of peroxide than originally observed for the mammalian enzyme (in the range of a few millimolar). This means that the limiting maximal turnover is less and the lifetime of the putative Michaelis-Menten intermediate (with the redox equivalent of two molecules of peroxide bound) is much longer. The extended scheme for catalase in Fig. 2B shows that relationships between free enzyme and compound I, and the presumed rate-limiting ternary complex with least stability or fastest decay in eukaryotic enzymes of type A and greatest stability or slowest decay in prokaryotic type B enzymes. 

Sirtuins have been conserved from bacteria to eukaryotes. Notably, they all possess a conserved catalytic core domain flanked by sequence-divergent N- and C-terminal regions. If bacteria and archaebacteria generally possess one or two sirtuins, this number is higher in eukaryotes, with five sirtuins in Saccharomyces cerevisiae and seven in human. The presence of sirtuins in all phyla of life led to a wealth of structural data, not only on eukaryotic enzymes but also on bacterial and archaebacteria enzymes. 

An important discovery is that procaryotes contain a different GTP cyclohydrolase 1 family distinct from the well studied canonical eukaryotic enzyme. Potentially, this enzyme is a target for new antibacterial drugs <2006JBC37586>. 

Hama, H. Almaula, N. Lerner, C.G. Inouye, S. Inoue, M. Nucleoside diphosphate kinase from Escherichia coli its overproduction and sequence comparison with eukaryotic enzymes. Gene, 105, 31-36 (1991)... 

Mitochondria contain a single RNA polymerase that resembles bacterial RNA polymerase more closely than it does the eukaryotic enzyme. 

A very different ribonuclease participates in the biosynthesis of all of the transfer RNAs of E. coli. Ribonuclease P cuts a 5 leader sequence from precursor RNAs to form the final 5 termini of the tRNAs. Sidney Altman and coworkers in 1980 showed that the enzyme consists of a 13.7-kDa protein together with a specific 377-nucleotide RNA component (designated Ml RNA) that is about five times more massive than the protein.779 Amazingly, the Ml RNA alone is able to catalyze the ribonuclease reaction with the proper substrate specificity.780 7823 The protein apparently accelerates the reaction only about twofold for some substrates but much more for certain natural substrates. The catalytic center is in the RNA, which functions well only in a high salt concentration. A major role of the small protein subunit may be to provide counterions to screen the negative charges on the RNA and permit rapid binding of substrate and release of products.783 Eukaryotes, as well as other prokaryotes, have enzymes similar to the E. coli RNase R However, the eukaryotic enzymes require the protein part as well as the RNA for activity.784... 

There is an absolute requirement for a DNA or RNA primer, which is annealed to the template and which possesses a 3 -hydroxyl group on the (deoxy)ribose. (Some very large eukaryotic enzymes have been found to have a primase activity by which they synthesize their own RNA primers.)... 

Enzymes that are structurally related to the eukaryotic V-ATPase are also found in certain eubacteria (Speelmans etal., 1994 Takase etal., 1994 Yokoyama etal., 1990). Based on nucleotide sequence analysis, it is believed that these bacterial V-like ATPases have been introduced into the eubacteria via horizontal gene transfer from Archaea (Hilario and Gogarten, 1993, 1998). The subunit composition of the bacterial V-like ATPase is indeed more similar to the archaeal A-ATPase than to the eukaryotic V-ATPase, and we will therefore treat the bacterial V-ATPase—like enzyme together with the archaeal A-ATPase (see below). In the following, we will use the name V-ATPase only for the eukaryotic enzyme, and we will call the bacterial enzyme the A/V-type ATPase as suggested by Hilario and Gogarten (1998). 

Each of the three eukaryotic RNA polymerases contains 12 or more subunits and so these are large complex enzymes. The genes encoding some of the subunits of each eukaryotic enzyme show DNA sequence similarities to genes encoding subunits of the core enzyme (a2PP ) of E. coli RNA polymerase (see Topic G2). However, four to seven other subunits of each eukaryotic RNA polymerase are unique in that they show no similarity either with bacterial RNA polymerase subunits or with the subunits of other eukaryotic RNA polymerases. 

The glycogen debranching enzyme is the first bifunctional eukaryotic enzyme to be reported that consists of a single polypeptide chain. It catalyzes two distinct activities an oligosaccharide frons-glycosylation followed by hydrolysis of an (1—> 6)-linked D-glucosyl unit to liberate free glucose. Physical-chemical and kinetic characterization of this novel bifunctional enzyme is described by Nelson. 

Based on the identified homology of the cefD and cefDZ proteins with known eukaryotic enzymes, a mechanism for the A. chrysogenum two-component epimerization system which is different from the epimerization found in prokaryotes has been established <2002JBC46216>. Therefore, it was suggested that the cephalosporin biosynthesis pathway begins with the activation of the substrate isopenicillin N to its CoA, followed by an epimerization to the D-enantiomer, namely penicillinyl-CoA. Next, the required hydrolysis of the CoA-thioesters seems to occur in a nonstereoselective manner by different thioesterases. The resulting product, penicillin N, is the direct precursor of all cephalosporins and cephamycins. 

Archaebacterial RNA polymerases are very different from their eubacterial counterparts and more closely resemble eukaryotic enzymes both in their subunit complexity and in their amino acid sequences (for review, see Puehler et al., 1989). This view is also reflected in the diversity of the DNA sequences that are used by the transcription apparatus as signals for initiation of transcription, namely, the promoters. Many attempts were made to identify a consensus promoter structure (Zillig et al., 1988). However, as more genes are isolated and characterized, the picture becomes less coherent. Earlier identification of two upstream sequences, box A and box B, located around positions — 30 and + 1, respectively, gave way to two elements —DPE (distal promoter element) and PPE (proximal promoter element)—located - 38 to - 25 and — 11 to — 2, respectively (Reiter et al., 1990). The DPE encompasses the box A sequence TTTA(A or T)A, but the PPE sequence seems to depend more on an (A + T)-rich sequence rather than on a specific DNA sequence. 

Very little is known about the structural domains that are responsible for the multiple functions of RNA polymerase. Therefore, comparison of the conserved amino acid sequences of the halobacterial and the eukaryotic enzymes might hint of the possible functional importance of these regions. For instance, the sequence alignment revealed that the zinc-binding motifs suggested for the eukaryotic enzymes are also conserved in the A and B subunits of the H. halobium enzyme, but only the former motif is partially conserved in the P subunit of the E. coli enzyme. Also, the splits of the B and the A subunits of the eukaryotic enzymes into two parts each in the halo-philic enzyme might suggest a division of these subunits into two functional domains. 

E. coli lipoamide dehydrogenase has a valine residue preceding the first half-cystine, whereas the eukaryote enzymes have a threonine residue. Chemically this is a relatively conservative change since the side chains of these amino acids are virtually identical in volume. 

Inositol is synthesized by the same route in all cells. D-Glucose-6-phosphate (d-G-6-P) is converted to L-mvo-inositol-1 -phosphate (l-I- 1 -P), which can also be termed D-myo-inositol-3-phosphate. The L-I-l-P is then hydrolyzed to myoinositol by a relatively specific phosphatase. As we will see, enzymes that do these reactions in bacteria and archaea can have quite different characteristics from the eukaryotic enzymes. 

NR activity is also sensitive to temperature, at least in diatom species. (Gao et al., 2000) demonstrated that NR activity in diatoms such as Skeletonema costatum tended to lose activity above 16°C, while other eukaryotic enzymes were stable at over 30° C. Lomas and Ghbert (1999) combined laboratory and field data to argue that this temperature sensitivity effectively Hmited diatoms to particular environments. Berges et al. (2002) speculated that features of the N-terminus of the NR protein might be responsible for temperature sensitivity, and these appear to be home out in data from recent sequencing projects (AUen et al., 2005). 

IC50 values were determined for selected compounds against the appropriate aaTRS from both spinach chloroplast and carrot cell cytoplasm (Table 2). No differentiation in potency was observed for inhibition of the chloroplastic, i.e. prokaryotic, and cytoplasmic, i.e. eukaryotic, enzymes. Enzymes from both tissues were equally susceptible to inhibition by the appropriate aminoacyl compound at nanomolar concentrations. Even closely related aminoacyl-sulfamoyladenosines were much weaker inhibitors, e.g. the leucyl derivative Ig and dealanylascamycin, la, were several orders of magnitude less potent than the appropriate aminoacyl-sulfamoyladenosines, compounds le and If, versus ITRS. 

Reversible covalent modification. The catalytic properties of many enzymes are markedly altered by the covalent attachment of a modifying group, most commonly a phosphoryl group. ATP serves as the phosphoryl donor in these reactions, which are catalyzed by protein kinases. The removal of phosphoryl groups by hydrolysis is catalyzed by protein phosphatases. This chapter considers the structure, specificity, and control of protein kinase A (PKA), a ubiquitous eukaryotic enzyme that regulates diverse target proteins. 

The unknotting reaction (typical of type II DNA topoisomerases) is ten times more efficient than relaxation. The most purified fraction contained two major polypeptides of 40 and 60 kDa. The S. acidocaldarius type II DNA topoisomerase has no DNA gyrase activity. This suggested that it could specifically resemble the eukaryotic type II DNA topoisomerase however, taking into account its putative dimeric structure, it could also resemble the new type II DNA topoisomerase (topo IV) recently discovered in E. co/i [70]. We have recently detected in our laboratory a type II DNA topoisomerase in Sulfolobus shibatae. This enzyme catalyzes the same reactions as the enzyme from Sulfolobus solfataricus and exhibits a pattern of drug sensitivity very similar to that of the eukaryotic enzyme [Bergerat, A., this laboratory]. 

FIGURE 17.2 Active site structures of the three families of mononuclear Mo- and W-enzymes. (a) Coordination around Mo. X and Y represent ligands such as oxygen (oxo, hydroxo, water, serine, aspartic acid), sulfur (cysteine), and selenium atoms (selenocysteine). (b) Structure of the pyranopterin molecule. R=H in eukaryotic enzymes and GMP, AMP, CMP or IMP in bacteria. Adapted from Brondino,... 

Over the past 30 years, a few researchers have reported the presence of xylose isomerase in a number of yeasts and fungi capable of rapid xylose metabohsm. Because of difficulties in using genetically engineered Saccharomyces, Freer et al. [38] re-examined Rhodosporidium toruloides to see if they could confirm an earlier report that this yeast produces xylose isomerase. They reasoned that the heterologous expression of an eukaryotic enzyme could fadfitate genetic engineering of xylose metabolism in S. cerevisiae. Unfortunately, they found that R. toruloides uses an oxidoreductase system fike other eukaryotes. Other approaches, however, have been more successful. 

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