Sulfhydryl enzymes, stabilizing

An enzyme similar to the 3 -nucleotidase of mung bean has been isolated from germinating wheat seedlings and purified 800-fold (90). The preparation possessed DNase, RNase, and 3 -nucleotidase activities. These three activities were similar in pH optima, requirements for Zn2+ and sulfhydryl compounds, stability to storage, temperature inactivation... 

Some of the basic information on stabilizing sulfhydryl enzymes, has been responsible for their commercialization. Without the judicious use of reducing compounds throughout the processing of the papaya latex, it would not have been possible to maximize the proteolytic activity of commercial papain preparations. Examples of other studies of enzymes which have contributed to commercialization are the determination of calcium ion as a requirement for amylase stability at high temperature, the difference in properties of catalases derived from bacterial, fungal, or... 

When a cell extract prepared from A. nicotianae FI1612 cells was stored without the addition of sulfhydryl-protecting reagents, 80% of the initial activity was lost after storage at 4°C for 4 days. The enzyme activity was stabilized... 

Ribonucleotide reductase is notable in that its reaction mechanism provides the best-characterized example of the involvement of free radicals in biochemical transformations, once thought to be rare in biological systems. The enzyme in E. coli and most eukaryotes is a dimer, with subunits designated R1 and R2 (Fig. 22-40). The R1 subunit contains two lands of regulatory sites, as described below. The two active sites of the enzyme are formed at the interface between the R1 and R2 subunits. At each active site, R1 contributes two sulfhydryl groups required for activity and R2 contributes a stable tyrosyl radical. The R2 subunit also has a binuclear iron (Fe3+) cofactor that helps generate and stabilize the tyrosyl radicals (Fig. 22-40). The tyrosyl radical is too far from the active site to interact directly with the site, but it generates another radical at the active site that functions in catalysis. 

MacDonald (99) showed that mouse liver acid phosphatase required active sulfhydryl groups for activity and that malonate buffer, pH 5.9, was useful for the assay of this enzyme because it stabilized the enzyme during the period of the assay. 

In a mechanism involving nucleophilic catalysis, the enzyme would promote nucleophilic attack on the carbonyl carbon to produce a tetrahedral intermediate. In this intermediate, resonance stabilization of the C-N bond has been destroyed and the barrier to rotation about the C-N bond greatly reduced. Collapse of the tetrahedral intermediate with expulsion of nucleophile can produce either the cis or trans Xaa-Pro peptide. In the original work on PPI (Fischer et ai, 1984), results were presented that indicated that an enzyme sulfhydryl group was required for activity. This result was later interpreted to support a mechanism involving nucleophilic catalysis (Fischer et ai, 1989a,b). 

The inhibition of hLAL by boronic acids and diethyl p-nitrophenyl phosphate (Sando and Rosenbaum, 1985 G. N. Sando and H. L. Brockman, unpublished, cited by Anderson and Sando, 1991) indicates that hLAL is a serine hydrolase. Two lipase/esterase consensus pentapep-tides, G-X-S-X-G, are found, but only one of them appears to be consistent with the packing requirements of the )8-eSer-a nucleophilic motif (see above). Susceptibility of the enzyme to sulfhydryl reagents, and the requirement of thiols for the stability of purified hLAL, prompted Anderson and Sando (1991) to propose that a cysteine residue, or rather a Cys/Ser couple, may be involved in an internal transacylation reaction. It must be pointed out, however, that hLAL has all three cysteines of the gastric enzyme (as well as six additional ones), and so the inhibitory Cys is also there. The same argument proposed herein with respect to hGL, i.e., that a free cysteine is topologically close to the active site, also holds for hLAL. 

Fig. 4. Ether phospholipid synthesis from dihydroxyacetone-phosphate. (A) Dihydroxyacetone-P acyl transferase (DHAPAT). The first step of ether phospholipid synthesis is catalyzed by peroxisomal DHAPAT. This enzyme is a required component of complex ether lipid biosynthesis and its role cannot be assumed by a cytosolic enzyme that also forms acyldihydroxyacetone-P. (B) Ether bond formation by alkyl-DHAP synthase. The reaction that forms the 0-alkyl bond is catalyzed by alkyl-DHAP synthase and is thought to proceed via a ping-pong mechanism. Upon binding of acyl-DHAP to the enzyme alkyl-DHAP synthase, the pro-f hydrogen at carbon atom 1 is exchanged by enolization of the ketone, followed by release of the acyl moiety to form an activated enzyme-DHAP complex. The carbon atom at the 1-position of DHAP in the enzyme complex is thought to carry a positive charge that may be stabilized by an essential sulfhydryl group of the enzyme thus, the incoming alkox-ide ion reacts with carbon atom 1 to form the ether bond of alkyl-DHAP. It has been proposed that a nucleophilic cofactor at the active site covalently binds the DHAP portion of the substrate.

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