Regulation of enzyme activity by enzyme orientation: A hypothesis

In addition to substrate binding sites, many enzymes must possess supersubstrate binding sites that regulate attachment and orientation of the enzyme toward the matrix (micelle, membrane) in which the substrate molecules are embedded, the supersubstrate.     

Mutual recognition between polymerized liposomes: enzyme and enzyme inhibitor system

In order to examine the usefulness of polymerized liposomes as a model for cell membranes, a mutual recognition phenomenon between different liposomes on which complementary ligands were attached was examined. We used trypsin- and soybean trypsin inhibitor (STI)-carrying polymerized liposomes to attain high sensitivities. The STI which was immobilized on the polymerized mono-dienoylphosphatidylcholine liposome showed a definite inhibitory effect on the catalytic activity of the trypsin which was immobilized on another polymerized liposome, whereas the inhibitory effect of the STI which was immobilized on the di-dienoylphosphatidylcholine liposome was much smaller than that of the mono-dienoylphosphatidylcholine system because of the larger rigity of the di-dienoylphosphatidylcholine liposome. These results suggest that the mutual recognition between complementary ligands can be realized by using polymerized liposomes with a physical stability and moderate deformability as their carriers.     

A counterintuitive approach to treat enzyme deficiencies: use of enzyme inhibitors for restoring mutant enzyme activity

Pharmacological chaperone therapy is an emerging counterintuitive approach to treat protein deficiencies resulting from mutations causing misfolded protein conformations. Active-site-specific chaperones (ASSCs) are enzyme active-site directed small molecule pharmacological chaperones that act as a folding template to assist protein folding of mutant proteins in the endoplasmic reticulum (ER). As a result, excessive degradation of mutant proteins in the ER-associated degradation (ERAD) machinery can be prevented, thus restoring enzyme activity. Lysosomal storage disorders (LSDs) are suitable candidates for ASSC treatment, as the levels of enzyme activity needed to prevent substrate storage are relatively low. In addition, ASSCs are orally active small molecules and have potential to gain access to most cell types to treat neuronopathic LSDs. Competitive enzyme inhibitors are effective ASSCs when they are used at sub-inhibitory concentrations. This whole new paradigm provides excellent opportunity for identifying specific drugs to treat a broad range of inherited disorders. This review describes protein misfolding as a pathophysiological cause in LSDs and provides an overview of recent advances in the development of pharmacological chaperone therapy for the diseases. In addition, a generalized guidance for the design and screening of ASSCs is also presented.     

The moderately efficient enzyme: evolutionary and physicochemical trends shaping enzyme parameters

The kinetic parameters of enzymes are key to understanding the rate and specificity of most biological processes. Although specific trends are frequently studied for individual enzymes, global trends are rarely addressed. We performed an analysis of k(cat) and K(M) values of several thousand enzymes collected from the literature. We found that the "average enzyme" exhibits a k(cat) of ~0 s(-1) and a k(cat)/K(M) of ~10(5) s(-1) M(-1), much below the diffusion limit and the characteristic textbook portrayal of kinetically superior enzymes. Why do most enzymes exhibit moderate catalytic efficiencies? Maximal rates may not evolve in cases where weaker selection pressures are expected. We find, for example, that enzymes operating in secondary metabolism are, on average, ~30-fold slower than those of central metabolism. We also find indications that the physicochemical properties of substrates affect the kinetic parameters. Specifically, low molecular mass and hydrophobicity appear to limit K(M) optimization. In accordance, substitution with phosphate, CoA, or other large modifiers considerably lowers the K(M) values of enzymes utilizing the substituted substrates. It therefore appears that both evolutionary selection pressures and physicochemical constraints shape the kinetic parameters of enzymes. It also seems likely that the catalytic efficiency of some enzymes toward their natural substrates could be increased in many cases by natural or laboratory evolution.     

Angiotensin converting enzyme: substrate inhibition

Phosphate, borate, and Tris inhibit angiotensin converting enzyme (ACE), but HEPES buffer is inert. Measurements of substrate inhibition were made in HEPES buffer at pH 7.0 and 25 degrees C and 37 degrees C. Substrate inhibition was marked and goes to completion. A new equation for substrate inhibitions enables one, under favorable circumstances, to determine whether there is cooperativity in the binding of substrate to the inhibitory and active sites. Cooperativity does occur with ACE using Hipp-His-Leu as substrate. The kinetic parameters were measured (Km = 0.21 mM, K* = 0.65 mM at 37 degrees C). The enzyme concentration (1.94 X 10(-8) M) was determined by titration with lisinopril so that kcat (5 X 10(3) at 37 degrees C) could be determined. Using this value and the molecular weight the specific activity of ACE was calculated for different common buffers. The specific activity in HEPES calculated from Vmax was 33.7 units/mg at 37 degrees C.     

Ribonuclease P: a ribonucleoprotein enzyme

The ribonucleoprotein ribonuclease P catalyzes the hydrolysis of a specific phosphodiester bond in precursor tRNA to form the mature 5' end of tRNA. Recent studies have shed light on the structures of RNase-P-RNA-P-protein and RNase-P-RNA-precursor-tRNA complexes, as well as on the positions of catalytic metal ions, emphasizing the importance of the structure to the catalytic function.     

REtools: A laboratory program for restriction enzyme work: enzyme selection and reaction condition assistance

Background  

Restriction enzymes are one of the everyday tools used in molecular biology. The continuously expanding panel of known restriction enzymes (several thousands) renders their optimal use virtually impossible without computerized assistance. Several manufacturers propose on-line sites that assist scientists in their restriction enzyme work, however, none of these sites meet all the actual needs of laboratory workers, and they do not take into account the enzymes actually present in one's own laboratory.     

Thermolysin: a peptide forming enzyme

Thermolysin, a thermostable endopeptidase, is recognised as a potential peptide bond forming enzyme. The importance of structural properties and its stereospecific nature towards peptide bond formation is described. Thermolysin's use in the keystep of the preparation of an artificial sweetener 'aspartame' is highlighted.