Class II enzymes

The fructose-specific PTS in R. sphaeroides is simpler than the one in E. coli or S. typhimurium in that it consists of only two proteins. Besides the fructose specific ll , a class II enzyme, there is only one cytoplasmic component called soluble factor (SF) [48]. We now know that SF consists of IIl , HPr and E-I covalently linked [109]. 11 and SF form a membrane-bound complex whose association-dissociation dynamics is much slower than the turnover of the system. Therefore, the complex is the actual catalytic unit in the overall reaction and P-enolpyruvate is the direct phosphoryl group donor [102],... 

There are two classes of aldolases. Class I aldolases, found in animals and plants, use the mechanism shown in Figure 14-5. Class II enzymes, in fungi and bacteria, do not form the Schiff base intermediate. Instead, a zinc ion at the active site is coordinated with the carbonyl oxygen at C-2 the Zn2+ polarizes the carbonyl group... 

MECHANISM FIGURE 27-14 Aminoacylation of tRNA by aminoacyl-tRNA synthetases. Step is formation of an aminoacyl adenylate, which remains bound to the active site. In the second step the aminoacyl group is transferred to the tRNA. The mechanism of this step is somewhat different for the two classes of aminoacyl-tRNA synthetases (see Table 27-7). For class I enzymes, (2a) the aminoacyl group is transferred initially to the 2 -hydroxyl group of the 3 -terminal A residue, then (3a) to the 3 -hydroxyl group by a transesterification reaction. For class II enzymes, ( the... 

In the two classes of synthetase the tRNAs approach the enzyme in a mirror-symmetric fashion. The 2 -OH of the terminal ribose is positioned to attack the carbonyl of the aminoacyl adenylate in class I enzymes, while the 3 -OH is positional for the attack in class II enzymes.207... 

Plants also produce structurally related enzymes (chitinases) that catalyse the hydrolysis of chitin (Table 12.2) and hence damage chitin-based insect integuments. Class I chitinases are basic enzymes with an jV-terminal hevein-related CBD and vacuole-targeting C-terminal signals whereas Class II enzymes are acidic proteins lacking these CBD and vacuole-targeting domains. Class IV chitinases are variously basic and acidic extracellular proteins with... 

A new subclass of enzymes that use SAM and an Fe4S4 center to generate a S -deoxyadenosyl radical is beginning to emerge. This subclass, designated as class III enzymes, is postulated to use SAM specifically as a cosubstrate rather than a cofactor as in the class I enzymes, or a cofactor generator as in the class II enzymes. The distinction is that in class III enzymes, SAM is stoichiometrically... 

CLASS II Enzyme only With tRNA With amino acid/ substrate analog Individual domains... 

Figure 3 Mode of tRNA binding to aaRSs. (a) Active site of GluRS enzyme bound to ATP and tRNA (PDB 1 N77). (b) Active site of AspRS enzyme bound to Asp-AMP and tRNA P (PDB 1 COA). Class I and Class II enzymes bind the tRNA acceptor stem from the minor and major groove sides, respectively. This orients either the 2 -OH or 3 -OH of A76 for specific attachment of the amino acid. ATP is represented by dark spheres, and tRNA is shown as a tube.

The overall two-step aminoacylation reaction relies on mechanistically distinct features of the Class I and Class II enzymes (1). In the Rossmaim fold of Class I aaRSs, ATP binding is stabilized by interactions with the conserved KMSKS and HIGH consensus sequences. The fi- and y-phosphates interact with Mg. The a-NH3+ group of the bound amino acid... 

Many methanogenic archaeabacteria lack cysteinyl-tRNA synthetase (CysRS). Interestingly, a Class II enzyme called O-phosphoseryl-tRNA synthetase (SepRS) acylates tRNA with O-phosphoserine (Sep) to form Sep-tRNA , which is then converted to Cys-tRNA by the enzyme Sep-tRNA Cys-tRNA synthase (SepCysS). It has been proposed that this indirect pathway may be the sole route for cysteine biosynthesis in these organisms (9). The crystal structure of SepRS was recently... 

At least one aminoacyl-tRNA synthetase exists for each amino acid. The diverse sizes, subunit composition, and sequences of these enzymes vv ere be vildering for many years. Could it be that essentially all synthetases evolved independently The determination of the three-dimensional structures of several synthetases follo ved by more-refined sequence comparisons revealed that different synthetases are, in fact, related. Specifically, synthetases fall into tvv o classes, termed class I and class II, each of vv hich includes enzymes specific for 10 of the 20 amino acids (Table 29.2). Glutaminyl-tRNA synthetase is a representative of class I. The activation domain for class I has a Rossmann fold (Section 16.1.101. Threonyl-tRNA synthetase (see Figure 29.11) is a representative of class II. The activation domain for class II consists largely of P strands. Intriguingly, synthetases from the tvv o classes bind to different faces of the tRNA molecule (Figure 29.14). The CCA arm of tRNA adopts different conformations to accommodate these interactions the arm is in the helical conformation observed for free tRNA (see Figures 29.5 and 29.6) for class II enzymes and in a hairpin conformation for class I enzymes. These two classes also differ in other ways. 

Class I enzymes acylate the 2 -hydroxyl group of the terminal adenosine of tRNA, whereas class II enzymes (except the enzyme for Phe-tRNA) acyl-ate the 3 -hydroxyl group. 

Most class I enzymes are monomeric, whereas most class II enzymes are dimeric. 

Why did two distinct classes of aminoacyl-tRNA synthetases evolve The observation that the two classes bind to distinct faces of tRNA suggests at least two possibilities. First, recognition sites on both faces of tRNA may have been required to allow the recognition of 20 different tRNAs. Second, it appears possible that, in some cases, a class I enzyme and a class II enzyme can bind to a tRNA molecule simultaneously without colliding with each other. In this way, enzymes from the two classes could work together to modify specific tRNA molecules. 

Class II enzymes are principally active against penicillins and are chromosomally-mediated. They are found in Proteus mirabilis and E. coli. 

Fig. 4. The hydrophobic channel of secretory phospholipases Aj. A schematic display looking direcdy into the channel showing the residues that comprise the mouth and internal contours m-l and sn-2 refer to the protruding termini of the alkyl substituents of the transition-state analog. The ellipses indicate residues whose side chains have been shown to be involved, or are likely to be involved, in interfacial adsorption. The hatched box corresponds to the location of the amino-terminal end of the catalytic network. Top The class I enzyme from Naja naja atra venom (White etal., 1990, copyrighted by the American Association for the Advancement of Science), middle the class II enzyme from human nonpancreatic sources (Scott et al., 1991), and bottom the class III enzyme from the venom of Apis melltfera, the European honey bee (Scott et al., 1990b).

Class I SPLA2S feature a distinctive loop of surface-exposed residues arising from the distal tip of the first antiparallel helix. This loop is absent in the class II enzymes, moderately developed among the class I elapids, and prominent among the class I enzymes from exocrine pancreas. 

Carboxyl-Terminal Extension of Class II Enzymes (Residues 126 to 134)... 

The carboxyl-terminal extension of class II enzymes forms a hemicir-cular bannister around the calcium-binding loop. It is secured proximally (Cys-126 = Cys-27) and distally (Cys-134 = Cys-50) by disulfide bridges. The 7- or 8-residue loop is rich in prolines and charged residues. This substructure is remote from the residues implicated in interfacial ad-sorpdon, substrate binding, and catalysis and has no defined catalytic or pharmacological role. 

Class II aldolases are normally dimeric, having a molecular weight for the subunit lying352 between 30,000 and 40,000. The Km (for D-fructose 1,6-bisphosphate) of these aldolases may be as high as 300 /xM, in contrast to 5 pM for Class I aldolases.352-354 The enzyme shows no activity toward D-fructose 1-phosphate. Class II enzymes are inhibited by EDTA and require Zn2+ or other metals for catalytic activity. The metal ion may involve polarization of the carbonyl group... 

Scheme 8 Two mechanistic proposals for the catalytic mechanism of CoA-transferases. In mechanism A, an acyl-enzyme Intermediate Is formed by reaction of an enzyme-bound glutamate (aspartate for Class III enzymes) with the donor acyl-CoA, followed by the formation of an enzyme-bound glutamyl- (or aspartyl-) CoA thioester Intermediate. The thioester subsequently reacts with the acceptor carboxylate to give a new acyl-enzyme anhydride from which the acyl group Is transferred to CoA. In Class I transferases, this process follows classical ping-pong kinetics, whereas In Class III enzymes the donor carboxylate only leaves the enzyme complex upon formation of the product (see text for details). Mechanism B represents a ternary complex mechanism as used by Class II enzymes In which a transient anhydride made up of the donor and acceptor acyl groups Is formed by reaction of the acceptor carboxylate with the donor acyl-ACP. The free ACP subsequently reacts with this anhydride to complete acyl transfer.

C-termini of CobL. Crystal structures of these enzymes of the anaerobic pathway revealed that both were methyltransferases, where the protein that aligns with the C-terminal region of CobL is a class II enzyme and not a member of the canonical B12 biosynthetic methyltransferase. The outcome of this is that the N-terminal region of CobL is likely to methylate precorrin-6B at C5 to give precorrin-7 and this is then methylated at C5 and decarboxylated to give precorrin-8. 

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