<p/>The pectin lyase activity in the commercial enzyme preparation Ultrazym originates from more then one type of enzyme; two of them, accounting for 95 % of the total activity, have been completely purified. As purity criteria specific activity, polyacrylamide disc gel electrophoresis and SDS electrophoresis have been used in combination.<p/>Both types of enzyme appear to be nearly identical with respect to their physico-chemical properties. The molecular weights, determined in different ways, and the iso-electric points are nearly identical, while the amino acid composition of both types shows a large degree of homology. The C-terminal amino acid residues are the same and the N-terminal amino acid residues are blocked in both cases; the enzymes are single chain proteins. In the fluorescence emission spectra of both types of enzyme a separate contribution of the tyrosine and tryptophan residues is seen; the quantum yield is not identical for both enzymes.<p/>The sugar composition is not completely identical for both types of enzyme; pectin lyase type I contains only mannose residues (4 moles/mole), whereas type II can be separated with affinity chromatography into two species which are only different in their ratio of mannose to glucose residues (mannose plus glucose is 4 moles/mole) but in no other respect.<p/>Pure pectin lyase type I is homogeneous upon polyacrylamide disc gel electrophoresis, whereas type II is always divided into two protein bands; the ratio between these bands depends on incubation conditions.<p/>At both sides of the pH range 4-6, over which the enzyme is stable, inactivation and denaturation occurs which is not completely reversible. The stability is largely influenced by the ionic strength; pectin lyase type II needs a higher ionic strength then type I for maintenance of the active conformation. The inactivation above pH 6 appears to be due to a combined effect of μand pH; this process is a first order equilibrium process, as determined for type I, of which the k <sub><font size="-1">1</font></sub> is the pH dependent and k <sub><font size="-1">-1</font></sub> the ionic strength dependent parameter. In the case of type II the conformational change upon inactivation is accompanied by a change in surface charge, resulting in two protein bands upon electrophoresis. For both, type I and type II, the inactivation is accompanied by (and can be followed by) a reduced contribution of tyrosine to the fluorescence of the protein.<p/>The three dimensional structure of the protein seems to be very rigid, as can be argued by the low α-helix content, the high molecular weights found by SDS electrophoresis, the two protein bands upon electrophoresis of type II in 8 M urea, the figures for the tyrosine content which are too low when determined by spectral analysis in 0.1 N NaOH and the lack of results upon modification of sulphydryl groups with Ellman's reagent under denaturating conditions.<p/>Studies on the kinetics of pectin lyase have been performed to obtain more insight in the action mechanism of this enzyme. The assignment of the influence of buffer (nature and concentration) and cations (nature and concentration) to an effect of the ionic strength and not to a specific ion effect explains the large variability in the reported kinetic characteristics and has enabled a detailed and accurate analysis of the influence of the pH on the kinetics.<p/>The most remarkable features of this analysis are: a bell shaped pH profile at low substrate concentrations, shifts of the pH optimum to more basic values upon increasing substrate concentrations,, accompanied by deviation of the profile from a bell shaped curve, at infinite substrate concentration a pH profile comparable with a saturation curve (with a slope not equal to a whole number in a Dixon plot), constant K <sub><font size="-1">m</font></sub> -values at low pH which increase at more basic values. An increase in the ionic strength results in a decrease in K <sub><font size="-1">m</font></sub> -values, most pronounced at high pH values, but do not affect the turnover numbers.<p/>These features can be explained by a model in which two ionic forms of the enzyme are able to bind substrate and to catalyze the reaction, although with different turnover numbers and binding constants. It could be concluded that the complexing of the enzyme with substrate has to occur by mediation of protonated groups. The pK values determined are assigned to carboxylic groups. This assignment is confirmed by the thermodynamic parameters of the ionization and by chemical modification. Besides carboxylic groups, one tyrosine residue is involved in the binding of the substrate as can be concluded from the pH profiles, chemical modification and quenching of the tyrosine fluorescence upon binding of substrate.<p/>From the temperature effects on the pH dependency of the kinetic parameters one may conclude that K <sub><font size="-1">1</font></sub> equals k <sub><font size="-1">cat</font></sub> /k <sub><font size="-1">1</font></sub> as is confirmed by the difference between the K <sub><font size="-1">m</font></sub> -value on pentamer and the K <sub><font size="-1">d</font></sub> of the enzyme with the same substrate as determined by fluorescence. Both enzymes behave similar with respect to those aspects just mentioned, only small differences in pK values and turnover numbers are found; the K <sub><font size="-1">m</font></sub> -values, and therefore also those of V/K <sub><font size="-1">m</font></sub> , on the contrary exhibits a pronounced difference.<p/>To obtain more insight in binding and catalysis of pectin lyase on a molecular level, the kinetics on oligomeric substrates have been studied and interpreted in terms of a subsite model. The number of subsites of pectin lyase type I amounts 9 - 10, whereas type II contains only 8 subsites. For the last enzyme the position of the catalytic place could be determined, whereas by using the reported K <sub><font size="-1">m</font></sub> -values of VORAGEN (1972) on substrates with different degree of esterification, that substrate could be determined which satisfies the demands for optimal binding. Moreover a method is given for evaluating the different subsite affinities by measuring directly dissociation constants of enzyme with oligomer.
|Qualification||Doctor of Philosophy|
|Award date||17 Oct 1975|
|Place of Publication||Wageningen|
|Publication status||Published - 1975|