Polypeptides from other enzymes

It is not easy to mimic the shuffling of domains in vitro by manipulation of genes. For example, each catalytic polypeptide chain of the multimeric E. coli aspartate transcarbamoylase (ATCase) is composed of two globular domains connected by two interdomain helixes. The E. coli enzyme ornithine transcarbamoylase (OTCase) is 32% identical in sequence and thus of presumably similar structure (see section D8). None of the chimeras in which a domain from one enzyme was attached to the corresponding partner in the other is active. The specific intrachain and interchain side-chain interactions also have to evolve for the Correcting packing.32... 

In prokaryotes, each of the reactions of fatty acid synthesis is catalyzed by a separate enzyme. However, in eukaryotes, the enzymes of the fatty acid synthesis elongation cycle are present in a single polypeptide chain, multifunctional enzyme complex, called fatty acid synthase. The fatty acid synthase complex exists as a dimer, with the ACP moiety shuttling the fatty acyl chain between successive catalytic sites, and from one subunit of the dimer to the other. It is, in effect, a highly efficient production line for fatty acid biosynthesis. 

Enzymes are proteins that catalyze reactions necessary to sustain life. Like other proteins, enzymes consist of chains of amino acids. The structural formulas of a few important amino acids are shown in Figure 29F-1. Molecules formed by linking two or more amino acids are called peptides. Each amino acid in a jjeptide is called a residue. Molecules with many amino acid linkages are polypeptides, and those with long polypeptide chains are proteins. Enzymes differ from other proteins in that a specific area of the structure, called the active site, assists in the catalysis. As a result, enzyme catalysis is often quite specific, favoring a particular substrate over other closely related compounds. 

Hydrochloric acid in the demineralizing compartment dissolves the bone mineral and denatures exposed collagen fibers for hydrolysis by cathepsin K. This lysosomal endopro-tease (Fig. 10.4a and b) has a cysteine thiol group at its catalytic center (Table 7.1 Fig. 10.4c). Cathepsin K is secreted in larger amounts than other lysosomal proteases and its specificity is due to unique amino acid residues around the peptide binding site. The enzyme hydrolyzes a) ) and a2(I) polypeptides from incompletely denatured fibers in the demineralizing compartment, leaving the cross-linked, telopeptide ends (Sect. 4.2.2). 

It has proved extremely useful for thrombin and many other enzymes to provide a standard nomenclature to describe the active site. The notation of Shechter and Berger is widely used for enzymes whose substrates are polymers the positions of the polymer are named -P4-P3-P2-P1/PT-P2 -P3 -P4 -, where / is the cleavage site, and the sequence for polypeptides runs from the N- to the C-terminus [12]. The corresponding pockets on the protein that are responsible for the recognition of these polymer elements are called sub-sites and are labeled. .. S2, SI, ST, S2. .. 

The presence of carbohydrate on protein molecules protects them from dena-turation. For example, bovine RNase A is more susceptible to heat denaturation than its glycosylated counterpart RNase B. Several other studies have shown that sugar-rich glycoproteins are relatively resistant to proteolysis (splitting of polypeptides by enzyme-catalyzed hydrolytic reactions). Because the carbohydrate is on the molecule s surface, it may shield the polypeptide chain from proteolytic enzymes. 

TTie key factor that sets enzymatic reactions apan from other catalyzed reactions is the formation of an enzyme-substrate complex, E S. Here substrate binds with a specific aaive site of the enzyme to form this complex. Figure 7-5 shows the schematic of the enzyme chymolrypsin (MW 25,000 Daltons), which catalyzes the hydrolytic clearage of polypeptide bonds. In many cases the enzyme s active catalytic sites are found where the various folds or loops interact. For chymoUy psin the catalytic sites are noted by the amino acid numbers 57, 102, and 195 in Figure 7-5. Much of the catalytic power is attributed... 

Many proteins that have the same type of function have similar protein sequences consequently, domains with similar conformations are associated with the particular function. Many types of domains have been identified, including three different types of domains by which proteins bind to DNA. In addition, short polypeptide sequences within a protein direct the posttransla-tional modification and subceUular localization. For example, several sequences play a role in the formation of glycoproteins (ones that contain sugars in addition to the polypeptide chain). Other specific sequences indicate that a protein is to be bound to a membrane or secreted from the cell. Still other specific sequences mark a protein for phosphorylation by a specific enzyme. 

HMG-CoA synthase (EC 4.1.3.5), catalyzing the formation of HMG-CoA and CoASH from acetyl-CoA and acetoacetyl-CoA, and not yet characterized from plants, was partially purified from C. roseus. It is an unstable enzyme that is rapidly inactivated in the presence of a relatively high concentration of salt (>200 mM). Thus far it has not been separated from AACT (81). HMG-CoA synthase activity elutes from gel filtration columns (e.g., Superose 6) with a retention time similar to that of polypeptides with an M, of about 100 kDa. HMG-CoA synthases isolated from other eukaryotic sources are mostly dimeric proteins of which the subunits have M, of 50-55 kDa for example, HMG-CoA synthase from chicken liver consists of two subunits of 53 kDa (82). When L-659,699, a /3-lactone inhibitor of mammalian cytosolic HMG-CoA synthase isolated from, among other sources, Fusarium sp. (83), is added to suspension cultured cells of... 

In the earlier experiments by Fischer and Bergell it was found that leucyl-alanine was hydrolysed by an extract of pancreas it was not however hydrolysed by pure pancreatic juice. Such extracts probably contain other enzymes, more especially the autolytic enzyme, which produce the hydrolysis the later work of Abderhalden and his coworkers upon the action of enzymes from various organs also show that polypeptides not hydrolysed by pure tiypsin are attacked by these enzymes (see table, p. 48). 

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