Enzyme substrate specificity

II. ENZYME SUBSTRATE SPECIFICITY CONSIDERATIONS IN DESIGN OF PRODRUGS... 

PK Banerjee, GL Amidon. Physicochemical property modification strategies based on enzyme substrate specificities I Rationale, synthesis, and pharmaceutical properties of aspirin derivatives. J Pharm Sci 70 1299, 1981. 

Kim, I., Song, X., Vig, B.S., Mittal, S., Shin, H.-C., Lorenzi, P.J. and Amidon, G.L., A novel nucleoside prodrug-activating enzyme substrate specificity of biphenyl hydrolase-like protein. Mol. Pharm., 2004,1, 117-127. 

The "lock-and-key" description of the catalytic action of enzymes given by Emil Fischer [13] one hundred years ago, put more emphasis on the enzyme-substrate specificity than on stereospecificity, suggesting the idea of ... 

The ability to biosynthetically incorporate noncoded amino acids into proteins site-specifically has facilitated studies not previously possible. These include studies of protein stability, the initiation of protein translation, electron transfer, protein-protein and protein-membrane interactions, reversal of enzyme substrate specificity, and structure-function relationships, among others. A growing number of research labs have begun to report applications of this technique. A brief look at some recent applications of the suppression mutagenesis technique follows. 

The properties and spatial arrangement of the amino acid residues forming the active site of an enzyme will determine which molecules can bind and be substrates for that enzyme. Substrate specificity is often determined by changes in relatively few amino acids in the active site. This is clearly seen in the three digestive enzymes trypsin, chymotrypsin and elastase (see Topic C5). These three enzymes belong to a family of enzymes called the serine proteases - serine because they have a serine residue in the active site that is critically involved in catalysis and proteases because they catalyze the hydrolysis of peptide bonds in proteins. The three enzymes cleave peptide bonds in protein substrates on the carboxyl side of certain amino acid residues. 

M. Wigger, J.P. Nawrocki, C.H. Watson, J.R. Eyler, S.A. Benner, Assessing enzyme substrate specificity using combinatorial libraries and ESI-FT-ICR-MS, Rapid Commun. Mass Spectrom., 11 (1997) 1749. 

Prior to the advent of recombinant DNA technology, the ability to design proteins was limited to chemical modification methods in which specific residues in a protein are modified at the protein level by chemical agents. Different strategies such as atom replacement and segment reassembly have been used to alter enzyme substrate specificity, activity, cofactor requirement, and stability.f These methods can introduce a diverse... 

A mutant enzyme with 17 amino acid substitutions was generated that shows a 2.1 x 10 -fold increase in the catalytic efficiency for a nonnative substrate, valine. The crystal structure of the mutant enzyme indicated a remodeled active site and altered subunit interface caused by the cumulative effects of mutations. Most amazingly, only one of the mutations directly contacts the substrate, which underscores our limited understanding of enzyme substrate specificity. These mutations would be difficult, if not impossible, to be identified and introduced to the mutant enzyme by a rational design approach. 

An impressive example is the creation of novel enzyme substrate specificity and activity by the DNA shuffling of two highly homologous triazine hydrolases, AtzA and TriA. These two enzymes catalyze the dechlorination and deamination reaction of atrazine and aminoatrazine, respectively. Although they share limited overlap in substrate preference, they only differ by 9 out of 475 amino acids. After one round of DNA shuffling, several variants were found to hydrolyze substrates that were not substrates for either of the parental enzymes. 

Huijghebaert et al. [23] isolated a bile salt sulfatase-producing strain designated, Clostridium S, from rat feces. This bacterium hydrolyzed the 3-sulfates of lithocholic acid, chenodeoxycholic acid, deoxycholic acid and cholic acid but not the 7-or 12-monosulfates. Sulfatase activity required the 3-sulfate group to be in the equatorial position. A free C-24 or C-26 carboxyl group was also required for sulfatase activity in whole cells of this bacterium. The 3-sulfate of cholesterol, Cj,-and Cji-steroids were not hydrolyzed by Clostridium S, [24]. Nevertheless, C,9- and C2]-steroid sulfates are hydrolyzed in the gut by microbial activity suggesting that the intestinal microflora may contain bacteria with steroid sulfatases possessing different substrate specificities. However, it should be noted that enzyme substrate specificity studies carried out in whole cells may reflect both cell wall permeability and enzyme specificity. 

Purification and characterizations of extracellular chitinases from the marine bacterium Bacillus sp. LJ-25 were described by Lee et al. (2000a). The purified chitinase so obtained showed a single band on SDS-PAGE and had an MW of approximately 50 kDa. The chitinase was most active and relatively stable at a pH of 7.0. The optimum temperature for this enzyme was around 35 °C when the pH of the reaction was kept at 7.0. The effect of metal ions on chitinase activity showed that Zn2+ strongly inhibited the enzyme activity. However, Ba2+, Co2+, Mn2+, and Cu2+ showed slight inhibition of the enzyme. Substrate specificity studies indicated that colloidal chitin (a substrate of the endo type of chitinase) was efficiently degraded by the chitinase. However, chitin and chitosan were ineffectively hydrolyzed by this enzyme. This chitinase did not hydrolyze /V,iV-diacetylchitobiose, j9-nitro phenol- /V- ace tyl - (3 -1 > - g I u c o s a mine, and Micrococcus lysodeikticus cells, which are known to be the substrates of the exo type of chitinases. 

The regulation of ribonucleotide reductase is quite complex. The enzyme contains two allosteric sites, one controlling the activity of the enzyme and the other controlling the substrate specificity of the enzyme. ATP bound to the activity site activates the enzyme dATP bound to this site inhibits the enzyme. Substrate specificity is more complex. ATP bound to the substrate site activates the reduction of pyrimidines (CDP and UDP), to form dCDP and dUDP. The dUDP is not used for DNA synthesis rather, it is used to produce dTMP (see below). Once dTMP is produced, it is phosphorylated to dTTP, which then binds to the substrate site and induces the reduction of GDP. As dGTP accumulates, it replaces dTTP in the substrate site and allows ADP to be reduced to dADP. This leads to the accumulation of dATP, which will inhibit the overall activity of the enzyme. These allosteric changes are summarized in Table 41.3. 

The AS bond appears to be of Importance for activity, perhaps due to enzymic substrate specificity, leading to a more metabollcally active compound. Such enzymic modification could be due to lipoxygenase or cyclooxygenase activity, which Is discussed In the final section of this review. 

N. Pi and J. A. Leary, Determination of enzyme/substrate specificity constant using multiple substrate ESI-MS assay, J. Am. Soc. Mass Spectrom. 15, 233-243 (2004). 

ESI Determination of enzyme/substrate specificity, kinetic evaluation Pi and Eeary [274]... 

Enzyme Substrate specificities Main source Remarks... 

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