Enzymatic hydrolysis of peptides containing ß-alanine
Creators: name: Hanson, H. Theo
Publisher: name: University of Utah
Year: 2012
Subjects: Analysis

description: An extensive review of the literature has revealed a dearth of information about the physiology and metabolism of B-alanine and the naturally occurring peptide, carnosine.  The widely-held belief that B-alanine compounds could not be split by proteolytic enzymes was based on very meager experimental evidence.  Our investigations conclusively show the fallacy of such generalization regarding proteases.  At the outset of our investigation of the metabolism of carnosine, it was demonstrated that extracts of several tissues rapidly hydrolyze carnosine.  The responsible enzyme from swine kidney has been purified fifteen-fold and some properties of the enzyme have been elucidated.  The enzyme was shown to be a metal-protein by the inhibition produced with such typical metal poisons as sulfide and cyanide.  Although both manganese and zinc ions activate carnosinase, it is likely that zinc in the metal which occurs in the enzyme as it is found it the tissue because of the similarity of the zinc-enzyme and the unactivated preparations with respect to (a) pH of optimal activity, (b) instability at 40? in the absence of substrate, and (c) insensitivity to phosphate and citrate ions.  For each of these properties, the unactivated enzyme resembles the zinc-enzyme rather than the preparation containing manganese.  Under the conditions of these experiments, the hydrolysis of carnosine is a zero order reaction, and the rate is proportional to the enzyme concentration over a reasonable range.  Evidence has been presented to show that the hydrolysis or carnosine cannot be attributed to my known protease.  Our present knowledge suggests that the hydrolysis of carnosine is due to a specific peptidase, carnosinase.  Wide-spread occurrence of an enzyme hydrolyzing carnosine probably accounts for the absence of this peptide from all tissues except muscle.  For the evaluation of the effect of the B-amino group on the specificity of several well-characterized peptidases, a large number of new peptides and derivatives of B-alanine were prepared.  The carbobenzoxy technique of Bergmann and Zervas proved eminently satisfactory for preparing these compounds.  Detailed direction for the syntheses and the physical constants of the compounds have been reported.  It has been conclusively demonstrated that the influence of the B-amino group of the substrate on its susceptibility to hydrolysis depends entirely upon the specificity of the individual enzyme.  For example, leucine aminopepidase of hog intestinal mucosa is unable to distinguish between and B and a B-amino group at the sensitive peptide bond; it hydrolyses L-leucyl- B-alanine at almost the same rate as L-leucylglylcine, one of the most sensitive substrates for this enzyme.   Crude leucine aminopeptidase preparations of human uterus and rat muscle also hydrolyze L-leucyl- B-alanine and show manganese activation.  With enzymes which require a free amino group adjacent to the sensitive peptide bond, such as prolidase, glycyl-L-leucine dipeptidase, and glycyclglycine dipeptidase, increasing the distance from the free amino group to the peptide bond decreases the sensitivity of the substrate quite markedly.  In most instance, it is possible to give a quantitative statement of this effect.  For example, the prolidases of hog intestinal mucosa and of rabbit muscle hydrolyze glycyl-L-proline about 200 to 300 time more rapidly B-alanyl-L-proline.  Moreover, the hydrolysis of glycyl-L-proline is inhibited about 50 percent in the presence of an equimolar concentration of the B-alanyl compound.  The hydrolysis of glycyl-L-leucine by glycyl-L-leucine dipeptidase from human uterus is about 200 times as fast as the of B-alanyl-L-leucine.   No hydrolysis or B-alanyl-B-alanine or of B-alanylglycine, and only slight hydrolysis of glycyl-B-alanine were detected, using preparations of human uterus or of rat muscle which are rich in glycylglycine dipeptidase.  Crystalline pancreatic carbroxypeptidase hydrolyzes carbobenzoxyglycylamino acids about 800 to 1600 times faster than the corresponding carbobenzoxy-B-alanylamino acids.  An extract of rat muscle containing a tripeptidase hydrolyzed triglycine, glycyl-B-alanylglycine, and diglycyl-B-alanine.  The action on B-alanylglycylglycine was a very slow, and was lost on standing under conditions which did not influence the splitting of other tripeptides.  It is evident tat the specificity of peptidase towards B-amino acids is not absolute, and B-alanine-containing peptides can be used to secure information regarding enzyme specificity.  It is apparent that the hydrolysis of compounds of unknown structure by proteolytic enzymes can no longer be regarded as proof the compound contains only B-amino acids.