Enzymes enzymatic activity

Enzyme Enzymatic activities (nkat/ml) Improvement of debarking... 

Km for an enzymatic reaction are of significant interest in the study of cellular chemistry. From equation 13.19 we see that Vmax provides a means for determining the rate constant 2- For enzymes that follow the mechanism shown in reaction 13.15, 2 is equivalent to the enzyme s turnover number, kcat- The turnover number is the maximum number of substrate molecules converted to product by a single active site on the enzyme, per unit time. Thus, the turnover number provides a direct indication of the catalytic efficiency of an enzyme s active site. The Michaelis constant, Km, is significant because it provides an estimate of the substrate s intracellular concentration. 

Use of ultrafiltration (UF) membranes is becoming increasingly popular for clarification of apple juice. AH particulate matter and cloud is removed, but enzymes pass through the membrane as part of the clarified juice. Thus pasteurization before UF treatment to inactivate enzymes prevents haze formation from enzymatic activity. Retention of flavor volatiles is lower than that using a rack-and-frame press, but higher than that using rotary vacuum precoat-filtration (21). 

It is essential to maintain high, maximal velocities of enzymatic activity for the attainment of optimal therapeutic efficacy. As a general rule, only enzymes whose MichaeHs-Menten constants He between 1—100 ]lM are effective as dmgs (16) because most substrates for therapeutically useful enzymes are present ia body fluids and cells at suhmillimolar concentrations. 

The transformations described thus far were catalyzed by enzymes in their traditional hydrolytic mode. More recent developments in the area of enzymatic catalysis in nonaqueous media (11,16,33—35) have significantly broadened the repertoire of hydrolytic enzymes. The acyl—enzyme intermediate formed in the first step of the reaction via acylation of the enzyme s active site nucleophile can be deacylated in the absence of water by a number of... 

Figure 4.7 Two of the enzymatic activities involved in the biosynthesis of tryptophan in E. coli, phosphoribosyl anthranilate (PRA) isomerase and indoleglycerol phosphate (IGP) synthase, are performed by two separate domains in the polypeptide chain of a bifunctional enzyme. Both these domains are a/p-barrel structures, oriented such that their active sites are on opposite sides of the molecule. The two catalytic reactions are therefore independent of each other. The diagram shows the IGP-synthase domain (residues 48-254) with dark colors and the PRA-isomerase domain with light colors. The a helices are sequentially labeled a-h in both barrel domains. Residue 255 (arrow) is the first residue of the second domain. (Adapted from J.P. Priestle et al., Proc.

The a/p-barrel structure is one of the largest and most regular of all domain structures, comprising about 250 amino acids. It has so far been found in more than 20 different proteins, with completely different amino acid sequences and different functions. They are all enzymes that are modeled on this common scaffold of eight parallel p strands surrounded by eight a helices. They all have their active sites in very similar positions, at the bottom of a funnel-shaped pocket created by the loops that connect the carboxy end of the p strands with the amino end of the a helices. The specific enzymatic activity is, in each case, determined by the lengths and amino acid sequences of these loop regions which do not contribute to the stability of the fold. 

Protein engineering is now routinely used to modify protein molecules either via site-directed mutagenesis or by combinatorial methods. Factors that are Important for the stability of proteins have been studied, such as stabilization of a helices and reducing the number of conformations in the unfolded state. Combinatorial methods produce a large number of random mutants from which those with the desired properties are selected in vitro using phage display. Specific enzyme inhibitors, increased enzymatic activity and agonists of receptor molecules are examples of successful use of this method. 

FIGURE 2.16 pH versus enzymatic activity. The activity of enzymes is very sensitive to pH. The pH optimum of an enzyme is one of its most important characteristics. Pepsin is a protein-digesting enzyme active in the gastric fluid. Trypsin is also a proteolytic enzyme, but it acts in the more alkaline milieu of the small intestine. Lysozyme digests the cell walls of bacteria it is found in tears. 

If the inhibitor combines irreversibly with the enzyme—for example, by covalent attachment—the kinetic pattern seen is like that of noncompetitive inhibition, because the net effect is a loss of active enzyme. Usually, this type of inhibition can be distinguished from the noncompetitive, reversible inhibition case since the reaction of I with E (and/or ES) is not instantaneous. Instead, there is a time-dependent decrease in enzymatic activity as E + I El proceeds, and the rate of this inactivation can be followed. Also, unlike reversible inhibitions, dilution or dialysis of the enzyme inhibitor solution does not dissociate the El complex and restore enzyme activity. 

The first hint that two active-site carboxyl groups—one proto-nated and one ionized—might be involved in the catalytic activity of the aspartic proteases came from studies of the pH dependence of enzymatic activity. If an ionizable group in an enzyme active site is essential for activity, a plot of enzyme activity versus pH may look like one of the plots at right. 

In die metabolic pathway to an amino add several steps are involved. Each step is die result of an enzymatic activity. The key enzymatic activity (usually die first enzyme in the synthesis) is regulated by one of its products (usually die end product, eg die amino add). If die concentration of die amino add is too high die enzymatic activity is decreased by interaction of die inhibitor with the regulatory site of die enzyme (allosteric enzyme). This phenomenon is called feedback inhibition. 

The reactivation of enzymes (after their partial inactivation in an acid medium) upon passing into a medium of pH 8 is also of great importance for oral use (Fig. 25). Enzymes immobilized in crosslinked polyelectrolytes are characterized by a structural memory even after considerable inactivation. Under changed conditions, this leads to a considerable or almost complete reactivation of the enzyme, whereas in the reactivation of a free enzyme in solution under similar conditions the enzymatic activity is restored on a lower level. 

Jervis used porous silica coated with chemisorbed polyacrylhydrazide for immobilization of adenosine monophosphate (AMP) [117]. After periodate oxidation of its ribose residue the ligand was coupled to the carrier and used for isolation of lactate dehydrogenase from rabbit muscle. The specific capacity was 2 mg of protein/g adsorbent with a ligand content of 10 pmol/g, whereas recovery of enzymatic activity after elution was 85%. Hipwell et al. [118] found that for effective binding of lactate dehydrogenases on AMP-o-aminoalkyl-Sepharose the spacer arm length required at least 4 methylene links. Apparently, a macromolecule of polyacrylhydrazide acts itself like an extended spacer arm and thus allow AMP to bind the enzyme. 

The enzymatic activity of these potentially harmful enzymes is tightly controlled. Once transcribed into protein, MMPs are expressed as inactive zymogens and require distinct activation processes to convert them into active enzymes. After secretion, MMP-activity is regulated by the noncovalent binding of tissue inhibitors of metalloproteinases ( TIMPs) as shown in Fig. 2 for MMP-2 and TIMP-2. Four TIMPs have been identified so far TIMP-1, TIMP-2, TIMP-3, and TIMP-4. All known MMPs can be inhibited by at least one of the four known TIMPs. Nevertheless, individual differences with regard to bond strength and thus the magnitude of inhibition of a particular MMP do exist. 

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