Methods in Molecular Biology • 16 Enzymes of Molecular Biology

CHAPTER 13

RNase A (EC 3.1.27.5)

Michael M. Burrell

1. Introduction
   The term ribonuclease (RNase) is an imprecise term and is used to
   cover both enzymes that cause exonucleolytic cleavage and endo-
   nucleolytic cleavage of RNA. Exonucleases may cleave the RNA in
   3'-5' direction or vice versa, whereas some endoribonucleases have a
   specific requirement for certain bases. For example the RNase from
   Bacillus cereus cleaves at pyrimidine residues (1). Some enzymes
   produce 5' phosphomononucleotides, whereas others give rise to 3'
   phosphomononucleotides. This chapter focuses on the endoribo-
   nuclease RNase A (otherwise described as RNase, RNase I, or pancre-
   atic ribonuclease), which shows some base specificity in where it
   cleaves RNA. The enzyme has been particularly well characterized at
   the molecular level.


2. The Enzyme
   The enzyme was obtained in crystalline form by Kunitz in 1940 (2)
   and the entire amino acid sequence of bovine pancreatic RNase A is
   now known. It has 124 amino acids and a mol mass of 12,600 Da (3).
   It is a fairly stable enzyme and contains four disulfide bridges, which
   occur in all mammalian pancreatic ribonucleases. When the bridges
   are reductively broken the protein is denatured and becomes inactive.
   On reoxidation the protein refolds and complete activity is restored
   (4). It is possible, however, to reduce the bridges only partially and
   retain enzyme activity. Removal of four peptides at the carboxyl termi-

From: Methods in Molecular Biology, VoL 16: Enzymes of Molecular Biology

Edited by: M. M. Burrell Copyright ©1993 Humana Press Inc., Totowa, NJ

263