Mutation of Enzymes

Lysine methyltransferases have been linked in various cases to the pathogenesis of cancer. Examples can be found for KMTs that induce repressive marks as well as for those that lead to an activating methylation pattern. Possible reasons for a disturbed regulation of methylation may result in mutations of enzymes or overexpression in cancer cells. An indication for a therapeutic benefit of potential inhibitors is often derived from siRNA knockdown of the requisite isotype. Mutations of MLLl for example are thought to be responsible for various forms of acute leukemia [63]. Overexpression of EZH2 was linked to breast or prostate cancer [64] and increased levels in human cancers have also been observed for G9a. Increased mRNA levels for... 

Point mutation of enzymes has played an important role in determining those amino acid residues involved in catalytic activities. It has also been used to improve the enantioselectivity of dehydrogenases. For example, even a single point mutation of a secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus can change substantially the enantioselectivity for the reduction of 2-butanone and 2-pentanone as shown in Table 15-6 45l... 

Keywords QCM Enzyme reaction DNA polymerase DNA hydrolysis DNA ligation DNA cleavage Glycosylation Phosphorylation Mutation of enzyme... 

E. T. Kaiser, D. S. Lawrence, Chemical mutation of enzyme active sites, Science, 1984, 226, 505-511. 

Interestingly, CPT does not bind to topoisomerase I and only weakly to B-DNA under physiological conditions [5], so both biomolecules and CPT are needed to form stable ternary complex 50. Additional evidence supporting such mechanism comes from the observation of resistance of cell lines toward CPT which have specific mutations of enzyme [180-182] (for medical consequences of CPT resistance, see [4, 183]). Similarly, deletion of gene coding topoisomerase I from yeast (Saccharomyces cerevisiae) resulted in fuU resistance of viable cells to CPT, with restoration of sensitivity to CPT after expression of human or yeast topoisomerase I [184, 185]. The schematic mechanism of CPT action is given in Fig. 22.6. 

On pharmacodynamic grounds, tumor resistance may be caused by such diverse mechanisms as the mutation or redundancy of topo II, the overexpression and preferred nuclear localization of proteasome a-type subunits (leading to a anomalous degradation of topo II), genetic deletion or loss-of-function mutations of p53, overexpression of ROS-detoxifying enzymes, overexpression of Bcl-2 (leading to a diminished cyt c release), etc. However, none of these factors would universally predict the development of anthracycline-resistance in a given tumor or another. 

The class A enzymes have Mx values around 30,000. Their substrate specificities are quite variable and a large number of enzymes have emerged in response to the selective pressure exerted by the sometimes abusive utilization of antibiotics. Some of these new enzymes are variants of previously known enzymes, with only a limited number of mutations (1 4) but a significantly broadened substrate spectrum while others exhibit significantly different sequences. The first category is exemplified by the numerous TEM variants whose activity can be extended to third and fourth generation cephalosporins and the second by the NMCA and SME enzymes which, in contrast to all other SXXK (3-lactamases, hydrolyse carbapenems with high efficiency. Despite these specificity differences, the tertiary structures of all class A (3-lactamases are nearly superimposable. 

In principle, numerous reports have detailed the possibility to modify an enzyme to carry out a different type of reaction than that of its attributed function, and the possibility to modify the cofactor of the enzyme has been well explored [8,10]. Recently, the possibility to directly observe reactions, normally not catalyzed by an enzyme when choosing a modified substrate, has been reported under the concept of catalytic promiscuity [9], a phenomenon that is believed to be involved in the appearance of new enzyme functions during the course of evolution [23]. A recent example of catalytic promiscuity of possible interest for novel biotransformations concerns the discovery that mutation of the nucleophilic serine residue in the active site of Candida antarctica lipase B produces a mutant (SerlOSAla) capable of efficiently catalyzing the Michael addition of acetyl acetone to methyl vinyl ketone [24]. The oxyanion hole is believed to be complex and activate the carbonyl group of the electrophile, while the histidine nucleophile takes care of generating the acetyl acetonate anion by deprotonation of the carbon (Figure 3.5). 

Initial approaches to directed evolution of enzymes rested upon the introduction of random mutations in random sites of the enzyme by the use of the error-prone PCR technique [92] or on the DNA-shuffling method [93]. Extensive research has also been reported in which every amino acid site in an enzyme was systematically subjected to saturation mutagenesis [94]. 

While many diseases have long been known to result from alterations in an individual s DNA, tools for the detection of genetic mutations have only recently become widely available. These techniques rely upon the catalytic efficiency and specificity of enzyme catalysts. For example, the polymerase chain reaction (PCR) relies upon the ability of enzymes to serve as catalytic amplifiers to analyze the DNA present in biologic and forensic samples. In the PCR technique, a thermostable DNA polymerase, directed by appropriate oligonucleotide primers, produces thousands of copies of a sample of DNA that was present initially at levels too low for direct detection. 

Mutation of the dihydrolipoate reductase component impairs decarboxylation of branched-chain a-keto acids, of pyruvate, and of a-ketoglutarate. In intermittent branched-chain ketonuria, the a-keto acid decarboxylase retains some activity, and symptoms occur later in life. The impaired enzyme in isovaleric acidemia is isovaleryl-CoA dehydrogenase (reaction 3, Figure 30-19). Vomiting, acidosis, and coma follow ingestion of excess protein. Accumulated... 

Two mechanisms of chromosomal resistance have been identified. A mutation of dihydropteroate synthetase (DHPS) in Strep, pneumoniae produces an altered enzyme with reduced affinity for sulphonamides. Hyperproduction of p-aminobenzoic acid (PABA) overcomes the block imposed by inhibition ofDHPS. The specific cause of PABA hyperproduction is unknown, though chromosomal mutation is the probable cause. 

As described above, simple mutation, regardless of rational or random, sometimes changes the function of enzymes in a drastic manner. Especially, in the case of enzymes belonging to enolase superfamily, including decarboxylases, consideration of the reaction mechanism is important because the apparently different transformations proceed via a similar key intermediate. Thus, the well-designed mutation and structure of the substrates will lead to a successful expansion of the application of enzymes in organic synthesis. 

A single mutation affects enzyme activity but not elicitorand PGIP binding abilities of endopolygalacturonase... 

---