The Ribonucleolytic Activity of Angiogenin

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Abstract
Angiogenin (ANG), a homologue of bovine pancreatic ribonuclease A (RNase A), promotes the growth of new blood vessels. The biological activity of ANG is dependent on its ribonucleolytic activity, which is far lower than that of RNase A. Here, the efficient heterologous production of human ANG in Escherichia coli was achieved by replacing two sequences of rare codons with codons favored by E. coli. Hypersensitive fluorogenic substrates were used to determine steady-state kinetic parameters for catalysis by ANG in continuous assays. The ANG pH−rate profile is a classic bell-shaped curve, with pK1 = 5.0 and pK2 = 7.0. The ribonucleolytic activity of ANG is highly sensitive to Na+ concentration. A decrease in Na+ concentration from 0.25 to 0.025 M causes a 170-fold increase in the value of kcat/KM. Likewise, the binding of ANG to a tetranucleotide substrate analogue is dependent on [Na+]. ANG cleaves a dinucleotide version of the fluorogenic substrates with a kcat/KM value of 61 M-1 s-1. When the substrate is extended from two nucleotides to four or six nucleotides, values of kcat/KM increase by 5- and 12-fold, respectively. Together, these data provide a thorough picture of substrate binding and turnover by ANG.