On the Mechanism of Formation of a Partially Active Aldolase by Tryptic Digestion

Abstract

The limited proteolysis of a fully active mercury derivative of rabbit muscle aldolase affects all four subunits of the enzyme, but not in a uniform way. At least four out of the nine peptides liberated are cleaved off from only two subunits. The specific activity of the product (aldolase-T) is 50°/, of that of the native enzyme. The combined kinetic and peptide analytical study of aldolase-T formation has shown that the diminished activity is the consequence of the splitting off of a particular tyrosine-containing peptide from all four subunits of aldolase, and of the cleavage of a peptide bond in the vicinity of residue Cys-341 in only two subunits. The data presented, together with the results of the polyacrylamide gel electrophoresis of aldolase-T in the absence and presence of sodium dodecylsulfate, support the idea that the structure of oligomeric aldolase is of type AABB. Large segments of one kind of the subunits can be split off without affecting the tetrameric structure. A tentative sequence of the C-terminal 60 amino acids of aldolase is also presented.According to our present knowledge rabbit muscle aldolase consists of four polypeptide chains of closely similar primary structure and the subunits operate independently of each other [i]. However, the investigations of the action of carboxypeptidase on the enzyme suggested that there were differences in the steric structure of the subunits [2-41. The tryptic digestion of native proteins proved to be a very sensitive method to reveal changes in the steric structure of enzymes following chemical modifications [5,6]. Therefore we undertook to study the structure of aldolase by tryptic digestion.We have shown previously that native aldolase is unsuitable for proteolytic investigations since in this case only large amounts of trypsin bring about detectable digestion and the protein is degraded with the parallel loss of enzymatic activity. We found however, a suitable object for the above type of studies in a fully active mercury derivative of aldolase, namely sldolase-(SHg),, in which two SH groups per subunit are blocked with p-mercuribenzoate. Although this modification induces changes in the steric structure of the protein, yet aldolase-(SHg), retains full enzymatic activity [7,8]. Addition of small amounts of trypsin to this mercury derivative of aldolase led to limited proteolysis, inasmuch as after splitting of about 24 peptide linkages per mole of aldolase the digestion stopped.Enzyme. Fructose-1,6-bisphosphate : D-glyceraldehyde-3-phosphate lyase (EC 4.1.2.13).

Crosslinking with Bifunctional Reagents as a Means for Studying the Symmetry of Oligomeric Proteins

“…Before use the enzyme solutions were freed from ammonium sulfate by gel filtration on a Sephadex G-50 column equilibrated with 0.2 M triethanolamine-HCI, pH 8.5. Protein concentration was determined on the basis of absorption coefficients = 0.74 for aldolase [16] and A%? = 1.29 for lactate dehydrogenase-M4 [17].…”

Amino acid sequence of the N‐terminal 158 residues of rabbit muscle aldolase

“…Another way of getting large fragments of the N-terminal portion of the molecule, which facilitated establishment of overlaps, was the limited tryptic digestion of PMB-treated aldolase. Under the experimental conditions applied a limited proteolytic process leads to the formation of a 50% active aldolase, denoted as aldolase-T [17,18]. During the course of this limited digestion two large peptides could be isolated which contained 27 and 41 amino acid residues from the N-terminal end of the protein [18].…”

“…Under the experimental conditions applied a limited proteolytic process leads to the formation of a 50% active aldolase, denoted as aldolase-T [17,18]. During the course of this limited digestion two large peptides could be isolated which contained 27 and 41 amino acid residues from the N-terminal end of the protein [18]. These peptides, called Nt-27 and Nt-41, contained peptides Tlb, T2a and T3 (Nt-27) and Tlb, T2a, T3 and T4a (Nt-41).…”

What’s in a name? From “fluctuation fit” to “conformational selection”: rediscovery of a concept

“…Szabolcsi continued her work, started in 1956, on the relationship between the structure and function of enzymes (Szabolcsi & Biszku, 1961 ). Her name is associated with the introduction of proteolysis (protein degradation catalysed by proteases) in a study of the fine structure of enzymes and proteins (Biszku et al, 1964 ), and she has experimentally demonstrated that certain structural elements of enzymes have motility. In 1963, she wrote a dissertation for the title “Doctor of the Biological Sciences” (see later) in which, in addition to her own results, she discussed in detail the enzymological problems of the age, including Koshland's induced fit model.…”