Residues Involved in the Catalysis, Base Specificity, and Cytotoxicity of Ribonuclease from Rana catesbeianaBased upon Mutagenesis and X-ray Crystallography

Abstract

The Rana catesbeiana (bullfrog) ribonucleases, which belong to the RNase A superfamily, exert cytotoxicity toward tumor cells. RC-RNase, the most active among frog ribonucleases, has a unique base preference for pyrimidine-guanine rather than pyrimidine-adenine in RNase A. Residues of RC-RNase involved in base specificity and catalytic activity were determined by sitedirected mutagenesis, k cat /K m analysis toward dinucleotides, and cleavage site analysis of RNA substrate. The results show that Pyr-1 (N-terminal pyroglutamate), Lys-9, and Asn-38 along with His-10, Lys-35, and His-103 are involved in catalytic activity, whereas Pyr-1, Thr-39, Thr-70, Lys-95, and Glu-97 are involved in base specificity. The cytotoxicity of RC-RNase is correlated, but not proportional to, its catalytic activity. The crystal structure of the RC-RNase⅐d(ACGA) complex was determined at 1.80 Å resolution. Residues Lys-9, His-10, Lys-35, and His-103 interacted directly with catalytic phosphate at the P 1 site, and Lys-9 was stabilized by hydrogen bonds contributed by Pyr-1, Tyr-28, and Asn-38. Thr-70 acts as a hydrogen bond donor for cytosine through Thr-39 and determines B 1 base specificity. Interestingly, Pyr-1 along with Lys-95 and Glu-97 form four hydrogen bonds with guanine at B 2 site and determine B 2 base specificity.Ribonucleases are found widely within living organisms and are thought to play an important role in the metabolism of RNA. Recently, it has been shown that several members of the bovine ribonuclease superfamily exhibit biological functions in addition to intrinsic ribonucleolytic activities. Human eosinophil-derived neurotoxin and eosinophil cationic protein exert neurotoxicity (1) as well as antiparasitic activity (2), human angiogenin induces blood vessel formation (3), and frog ribonuclease exhibits antitumor activity (4, 5). Ribonucleolytic activity is essential for the biological functions of these proteins (6 -12).Bovine pancreatic ribonuclease, known as RNase A, in the ribonuclease superfamily is well characterized and is a valuable model for the study of structure-function relationships and protein refolding (13,14). It consists of 124 amino acid residues linked with four pairs of disulfide bridges and possesses a substrate preference for pyrimidine-adenosine in the RNA sequence but no cytotoxicity toward tumor cells. There are three subsites within RNase A molecule: the P 1 site, at which phosphodiester bond cleavage occurs; the B 1 site, for binding pyrimidine, which donates oxygen via its ribose to the scissile bond; and the B 2 site, for binding the adenine ring on the opposite site of the scissile bond. Three amino acid residues, His-12, Lys-41, and His-119, at the P 1 site are involved in catalytic activity. Four amino acid residues, Thr-45, Asp-83, Phe-120, and Ser-123, at the B 1 site are involved in the binding of the 5Ј-ribonucleoside, pyrimidine, whereas two residues, Asn-71 and Glu-111, at the B 2 site are involved in the binding of the 3Ј-ribonucleoside, adenosine (14 -18).A new group of ribon...

Transfer RNA Cleavages by Onconase Reveal Unusual Cleavage Sites

“…At increasing enzyme concentrations, additional cleavage products, species A, E, and I, were also seen. Species A, I, and E resulted from cleavages 3Ј to pyrimidine as observed for onconase with synthetic substrates (23)(24)(25)(26). Other cleavages seen were less intense.…”

“…Other cleavages were apparent as the enzyme concentration increased. Earlier studies on onconase action on synthetic substrates (23)(24)(25)(26) have suggested a preference for UpG (U binding in the B1 sub-site and G in the B2 sub-site of the substrate-binding site of enzyme). Out of the intense cleavages observed, species A resulted from the cleavage 3Ј to pyrimidine within the dinucleotide U-G. Cleavage at U-G, generating species A, and other cleavages such as those involving residues C-G, are consistent with the base specificity already reported for onconase.…”

“…However, the basis of this specificity toward tRNA has not been understood. When tried with synthetic substrates, onconase predominantly cleaved dinucleotides UpG and CpG (22)(23)(24)(25)(26). Our present studies indicate that the cleavages by onconase in several tRNAs involve selective G-G sites in addition to U-G and C-G dinucleotide bonds.…”

The Putative Transmembrane Segment 7 of Human Organic Anion Transporter hOAT1 Dictates Transporter Substrate Binding and Stability

“…In contrast, substitution of Tyr-354 with either Phe or Trp could not recover the transport activity lost by substitution with alanine, suggesting that both the ϪOH group and the size of the side chain at position 353 and 354 are essential for the transport function. The ϪOH group often participates in substrate binding or maintaining the protein structure through forming hydrogen bonds (DiezSampedro et al, 2001;Witt et al, 2002;Leu et al, 2003).…”

Structure–activity relationships in Kluyveromyces lactis γ-toxin, a eukaryal tRNA anticodon nuclease

“…(A glutamine side chain in RNase A also donates a hydrogen bond to the scissile phosphodiester.) By contrast, the RNase A-like Rana catesbeiana ribotoxin uses two lysines to coordinate the scissile phosphodiester (Leu et al 2003). Lysine can also serve as a general acid to expel a 59-OH polynucleotide leaving group, for example, during the DNA cleavage transesterification reaction catalyzed by topoisomerase IB (Krogh and Shuman 2000).…”