Biochemical and genetic analysis of a maltopentaose-producing amylase from an alkaliphilic gram-positive bacterium.

Two amylases have been purified from the culture fluid of an alkaliphilic bacterium. Amylase A-60 consists of a single type of polypeptide chain of 60 kDa and exhibits an alpha-amylase-type of starch cleavage. Amylase A-180 is approximately 180 kDa in size, represents the largest exoenzyme so far identified in prokaryotes and in the initial enzyme reaction cleaves starch exclusively to maltopentaose. A-60 and A-180 are immunologically unrelated enzymes. The structural gene for amylase A-180 has been cloned and its nucleotide sequence was determined. An open reading frame was identified for a putative protein of 182 kDa whose amino-terminal sequence, deduced from the nucleotide sequence, was identical in 23 out of 25 positions to that determined for the protein. The amino-terminus of the mature protein, at the gene level, is preceded by a sequence segment showing all the characteristics of a signal peptide from Gram-positive bacteria. Analysis of the deduced amino acid sequence revealed that the 70-kDa N-terminal part is similar to classical alpha-amylases. The C-terminal part contains three repeated sequence blocks of 99 amino acid residues each which are also present in two bacterial beta-amylases. It appears, therefore, that A-180 has arisen by gene fusion events.     

Characterization of the Butyrivibrio fibrisolvens glgB gene, which encodes a glycogen-branching enzyme with starch-clearing activity.

A Butyrivibrio fibrisolvens H17c glgB gene, was isolated by direct selection for colonies that produced clearing on starch azure plates. The gene was expressed in Escherichia coli from its own promoter. The glgB gene consisted of an open reading frame of 1,920 bp encoding a protein of 639 amino acids (calculated Mr, 73,875) with 46 to 50% sequence homology with other branching enzymes. A limited region of 12 amino acids showed sequence similarity to amylases and glucanotransferases. The B. fibrisolvens branching enzyme was not able to hydrolyze starch but stimulated phosphorylase alpha-mediated incorporation of glucose into alpha-1,4-glucan polymer 13.4-fold. The branching enzyme was purified to homogeneity by a simple two-step procedure; N-terminal sequence and amino acid composition determinations confirmed the deduced translational start and amino acid sequence of the open reading frame. The enzymatic properties of the purified enzyme were investigated. The enzyme transferred chains of 5 to 10 (optimum, 7) glucose units, using amylose and amylopetin as substrates, to produce a highly branched polymer.     

Folding competence of N-terminally truncated forms of human procathepsin B

Besides acting as an inhibitor, the propeptide of human cathepsin B exerts an important auxiliary function as a chaperone in promoting correct protein folding. To explore the ability of N-terminally truncated forms of procathepsin B to fold into enzymatically active proteins, we produced procathepsin B variants progressively lacking N-terminal structural elements in baculovirus-infected insect cells. N-terminal truncation of the propeptide by up to 22 amino acids did not impair the production of activable procathepsin B. Secreted forms lacking the first 20, 21, or 22 amino acids spontaneously generated mature cathepsin B through autocatalytic processing, demonstrating that the first alpha-helix (Asp11-Arg20) is necessary for efficient inhibition of the enzyme by its propeptide. In contrast, proenzymes lacking the N-terminal part including the first beta-sheet (Trp24-Ala26) of the propeptide or containing an amino acid mutation directly preceding this beta-sheet were no longer properly folded. This shows that interactions between Trp24 of the propeptide and Tyr183, Tyr188, and Phe180 of the mature enzyme are important for stabilization and essential for procathepsin B folding. Thus, proenzyme forms missing more than the N-terminal 22 amino acids of the propeptide (notably truncated cathepsin B produced by the mRNA splice variant lacking exons 2 and 3, resulting in a propeptide shortened by 34 amino acids) are devoid of proteolytic activity because they cannot fold correctly. Thus, any pathophysiological involvement of truncated cathepsin B must be ascribed to properties other than proteolysis.     

Cloning, nucleotide sequence, and expression of Achromobacter protease I gene

Achromobacter protease I (API) is a lysine-specific serine protease which hydrolyzes specifically the lysyl peptide bond. A gene coding for API was cloned from Achromobacter lyticus M497-1. Nucleotide sequence of the cloned DNA fragment revealed that the gene coded for a single polypeptide chain of 653 amino acids. The N-terminal 205 amino acids, including signal peptide and the threonine/serine-rich C-terminal 180 amino acids are flanking the 268 amino acid-mature protein which was identified by protein sequencing. Escherichia coli carrying a plasmid containing the cloned API gene overproduced and secreted a protein of Mr 50,000 (API') into the periplasm. This protein exhibited a distinct endopeptidase activity specific for lysyl bonds as well. The N-terminal amino acid sequence of API' was the same as mature API, suggesting that the enzyme retained the C-terminal extended peptide chain. The present experiments indicate that API, an extracellular protease produced by gram-negative bacteria, is synthesized in vivo as a precursor protein bearing long extended peptide chains at both N and C termini.     

The region of the IncN plasmid R46 coding for resistance to beta-lactam antibiotics, streptomycin/spectinomycin and sulphonamides is closely related to antibiotic resistance segments found in IncW plasmids and in Tn21-like transposons.

The nucleotide sequence of a 2.5 kb segment of the pKM101 (R46) genome has been determined. The 1.3 kb from a BamHI site at 153 to base 1440 differs by only 2 bases from a part of the published sequence of the aadB (gentamicin resistance) gene region including the coding region for the N-terminal 70 amino acids of the predicted aadB product. The same sequence has been found 5'-to the dhfrII gene of R388 and to the aadA gene of Tn21 (R538-1). Three open reading frames are located in this region, two on the same strand as the resistance genes and one on the complementary strand. The latter predicts a polypeptide of 337 amino acids, whose N-terminal segment is 40% homologous to the predicted product of an open reading frame of 179 amino acids located next to the dhfrI gene of Tn7. The oxa2 (oxacillin resistance) gene predicts a long polypeptide commencing with (the N-terminal) 70 amino acids of the aadB product. A similar arrangement is found in the aadA gene of R538-1. The N-terminal segment of an aadA gene is located 3'- to oxa2, separated by 36 bases. Sequences surrounding the BamHI site are identical to sequences 5'- to the tnpM gene of Tn21 and homology ceases where homology between Tn21 and Tn501 commences. The possibility that this antibiotic resistance segment is a discrete mobile DNA element is discussed.     

Isolation of the structural gene encoding a mutant form of Escherichia coli phosphoenolpyruvate carboxylase deficient in regulation by fructose 1,6-bisphosphate. Identification of an amino acid substitution in the mutant

The structural gene encoding a mutant Escherichia coli phosphoenolpyruvate carboxylase deficient in regulation by fructose 1,6-bisphosphate (Fru-P2) was isolated from total E. coli PpcI genomic DNA. This mutant gene is located on a 4.4-kilobase SalI DNA fragment which, when ligated to SalI-digested pBR322, resulted in the generation of the plasmid pFS16. Detailed restriction mapping of the wild-type and mutant genes for phosphoenolpyruvate carboxylase revealed the presence of a ClaI restriction site at position 563 of the mutant gene only. This ClaI site is located on a 289 PvuII/DdeI fragment which codes for amino acid residues 174-270 of the phosphoenolpyruvate carboxylase enzyme. When this portion of the mutant gene is present in chimeras of the wild-type and mutant genes, the phosphoenolpyruvate carboxylase produced cannot be activated by Fru-P2. The mutation resulting in the generation of the ClaI site in the mutant gene has also resulted in an amino acid substitution at residue 188; threonine in the wild-type enzyme has been replaced by isoleucine in the mutant enzyme. Comparison of the nucleotide sequence of this 289-base pair PvuII/DdeI region of the mutant gene with its homologous region in the wild-type gene verified that this mutation, which resulted in the generation of the ClaI site, is the only change that has occurred on this 289-base pair fragment of the mutant gene, and thus the amino acid replacement of threonine by isoleucine is the only change that could be linked to the inability of the mutant enzyme to be activated by Fru-P2.     

亚位点?7处突变对碱性芽胞杆菌CGT酶产物特异性的影响

为了研究来源于碱性芽胞杆菌的γ-环糊精葡萄糖基转移酶(CGT酶)具有较高产物特异性的作用机理,对其氨基酸序列和模拟结构进行了分析,确定其亚位点7处氨基酸的缺失可能影响其产物特异性。运用重叠PCR的方法,在其亚位点7处添加缺失的6个氨基酸,造成插入突变。将突变基因与pET-20b(+)连接并在大肠杆菌BL21(DE3)中表达。以可溶性淀粉为底物进行酶转化,HPLC分析转化产物中的环糊精含量。结果表明,相对于野生型γ-CGT酶,突变酶转化生成的3种环糊精中,γ-环糊精所占的比例从76.0%降至12.5%,α-、β-环糊精分别从8.7%和15.2%提高至37.5%和50%。分析其可能机理为:与α-、β-CGT酶相比,野生型γ-CGT酶的亚位点7处缺失6个氨基酸,该构象为葡萄糖的结合提供了更大的空间,从而更适合γ-环糊精的生成;而在其亚位点7处插入6个氨基酸,造成插入突变后,葡萄糖链结合的空间变小,这种构象不利于γ-环糊精的生成。     

Role of aspartate-37 in determining cofactor specificity and binding in rat liver dihydropteridine reductase

Full-length rat dihydropteridine reductase (DHPR) cDNAs have been combined with a prokaryotic expression vector and introduced into Escherichia coli. Transformed bacteria express dihydropteridine reductase immunoreactive proteins and demonstrate conversion of quinonoid dihydropteridines to their tetrahydro forms. Several recombinant enzymes have been purified to homogeneity and biochemical studies have been carried out comparing their properties with those exhibited by the rat liver enzyme. The optimal reaction conditions, kinetic constants, and stability are similar for the recombinant and naturally occurring enzyme. The results indicate that the nonmutant recombinant rat DHPR is an authentic replica of the natural protein and that the characteristics of DHPR activity are determined by a single gene product and do not require specific modification via the eukaryotic cell. In addition to the wild type, three specific mutagenic forms of the reductase, A-6-V, W-104-F, and D-37-I, and an additional abbreviated structure have also been formed. Each of the products exhibits reductase activity, although they show varied affinities for their cofactor, NADH, and less stability to chromatography, dialysis, and concentration than the wild-type enzyme. The N-terminal sequence contains a classic NADH binding region between amino acids 9 and 36, and Asp 37 is essential for binding the cofactor as is shown by the approximately 20-fold increase in dissociation constant for the D-37-I mutant and diminished kcat (approximately 43 s-1 compared to 156 s-1 for the wild-type enzyme). The results indicate that the DHPR cofactor binding site is similar to typical dinucleotide requiring dehydrogenases such as lactic acid and liver alcohol dehydrogenase.     

Effect of deletions within the leader peptide of pre-ornithine transcarbamylase on mitochondrial import

The uptake of the cytoplasmically synthesized mammalian enzyme, ornithine transcarbamylase, into mitochondria is directed by an N-terminal peptide of 32 amino acids. We have investigated some of the structural requirements for the import of the enzyme from rat liver into isolated mitochondria and into mitochondria of COS cells transfected with cDNA encoding the precursor form of ornithine transcarbamylase. Deletion of 21 amino acids from the N terminus of the leader peptide blocked the import of the precursor; deletion of 5 amino acids at positions 15-19 from the N terminus of the leader peptide had no deleterious effect on the import of the enzyme, nor on the processing and assembly of subunits in mitochondria. The region deleted contained three of eight basic residues in the leader peptide suggesting that specific structural elements containing basic residues, rather than the general basic nature of the leader, may be involved in mitochondrial import.     

Cloning, phenotypic expression, and DNA sequence of the gene for lactacin F, an antimicrobial peptide produced by Lactobacillus spp

Lactacin F is a heat-stable bacteriocin produced by Lactobacillus acidophilus 11088. A 63-mer oligonucleotide probe deduced from the N-terminal lactacin F amino acid sequence was used to clone the putative laf structural gene from plasmid DNA of a lactacin F-producing transconjugant, L. acidophilus T143. One clone, NCK360, harbored a recombinant plasmid, pTRK160, which contained a 2.2-kb EcoRI fragment of the size expected from hybridization experiments. An Escherichia coli-L. acidophilus shuttle vector was constructed, and a subclone (pTRK162) containing the 2.2-kb EcoRI fragment was introduced by electroporation into two lactacin F-negative strains, L. acidophilus 89 and 88-C. Lactobacillus transformants containing pTRK162 expressed lactacin F activity and immunity. Bacteriocin produced by the transformants exhibited an inhibitory spectrum and heat stability identical to those of the wild-type bacteriocin. An 873-bp region of the 2.2-kb fragment was sequenced by using a 20-mer degenerate lactacin F-specific primer to initiate sequencing from within the lactacin F structural gene. Analysis of the resulting sequence identified an open reading frame which could encode a protein of 75 amino acids. The 25 N-terminal amino acids for lactacin F were identified within the open reading frame along with an N-terminal extension, possibly a signal sequence. The lactacin F N-terminal sequence, through the remainder of the open reading frame (57 amino acids; 6.3 kDa), correlated extremely well with composition analyses of purified lactacin F which also predicted a size of 51 to 56 amino acid residues. Molecular characterization of lactacin F identified a small hydrophobic peptide that may be representative of a common bacteriocin class in lactic acid bacteria.     

Purification, characterization, gene cloning, sequencing, and overexpression of aminopeptidase N from Streptococcus thermophilus A.

The general aminopeptidase PepN from Streptococcus thermophilus A was purified to protein homogeneity by hydroxyapatite, anion-exchange, and gel filtration chromatographies. The PepN enzyme was estimated to be a monomer of 95 kDa, with maximal activity on N-Lys-7-amino-4-methylcoumarin at pH 7 and 37 degrees C. It was strongly inhibited by metal chelating agents, suggesting that it is a metallopeptidase. The activity was greatly restored by the bivalent cations Co2+, Zn2+, and Mn2+. Except for proline, glycine, and acidic amino acid residues, PepN has a broad specificity on the N-terminal amino acid of small peptides, but no significant endopeptidase activity has been detected. The N-terminal and short internal amino acid sequences of purified PepN were determined. By using synthetic primers and a battery of PCR techniques, the pepN gene was amplified, subcloned, and further sequenced, revealing an open reading frame of 2,541 nucleotides encoding a protein of 847 amino acids with a molecular weight of 96,252. Amino acid sequence analysis of the pepN gene translation product shows high homology with other PepN enzymes from lactic acid bacteria and exhibits the signature sequence of the zinc metallopeptidase family. The pepN gene was cloned in a T7 promoter-based expression plasmid and the 452-fold overproduced PepN enzyme was purified to homogeneity from the periplasmic extract of the host Escherichia coli strain. The overproduced enzyme showed the same catalytic characteristics as the wild-type enzyme.     

Effects of N-Terminal Deletion Mutation on Rabbit Muscle Lactate Dehydrogenase

Deletion mutants of rabbit muscle lactate dehydrogenase (LDH) were constructed using polymerase chain reaction (PCR) to study the roles of N-terminal residues. The coding sequences of the first 5 (LD5) and 10 (LD10) amino acids of the N-terminus were deleted and the gene was inserted into the prokaryotic expression vector pET21b. The mutant enzymes were expressed in E. coli BL21/DE3 and were purified. Then their characteristics and stabilities were studied. The results showed LDH was completely inactivated when the first 10 N-terminal amino acid residues were removed, but the mutant (LD10) could have partially restored activity in the presence of structure-making ions. The removal of the first 5 and 10 N-terminal amino acid residues did not affect the aggregation state of the enzyme, that is, LD5 and LD10 were still tetramers. The stabilities of recombinant wild-type LDH (RW-LD), LD5, and LD10 were compared by incubating them at low pH, elevated temperature, and high GuHCl. The results showed that the N-terminal deletion mutants were more sensitive to denaturing environments; they were easily inactivated and unfolded. Their instability increased and their ability to refold decreased with the increased number of amino acid residues removed from the N-terminus of LDH. These results confirm that the N-terminus of LDH plays a crucial role in stabilizing the structure and in maintaining the function of the enzyme.     

The 15 N-terminal amino acids of hexokinase II are not required for in vivo function: analysis of a truncated form of hexokinase II in Saccharomyces cerevisiae

The function of the N-terminal amino acids of Saccharomyces cerevisiae hexokinase II was studied in vivo using strains producing a form of hexokinase II lacking its first 15 amino acids (short form). This short form of hexokinase II was produced from a fusion between the promoter region of the PGK1 gene and the HXK2 coding sequence except the first 15 codons. As expected, the in vitro analysis of the short form protein by gel filtration chromatography indicates that the short protein does not form dimers under conditions where the wild-type protein dimerizes. Kinetic studies show that the enzymatic activities are very similar to wild-type behavior. The physiological experiments performed on the strains containing the fusion allele demonstrate that the short form of the enzyme is similar to the wild-type both in terms of phosphorylation of hexoses and glucose repression. We conclude that the N-terminal amino acids of hexokinase II are not required in vivo either for phosphorylation of hexoses or for glucose repression.     

Molecular dynamics simulation studies of a protein-RNA complex with a selectively modified binding interface

The RNA recognition motif (RRM) is one of the most common RNA binding domains. We have investigated the contribution of three highly conserved aromatic amino acids to RNA binding by the N-terminal RRM of the U1A protein. Recently, we synthesized a modified base (A-4CPh) in which a phenyl group is tethered to adenine using a linker of 4 methylene groups. The substitution of this base for adenine in the target RNA selectively stabilizes the complex formed with a U1A protein in which one of the conserved aromatic amino acids is replaced with Ala (Phe56Ala). In this article, we report molecular dynamics (MD) simulations that probe the structural consequences of the substitution of A-4CPh for adenine in the wild type and Phe56Ala U1A-RNA complexes and in the free RNA. The simulations suggest that A-4CPh stabilizes the complex formed with Phe56Ala by adopting a folded conformation in which the tethered phenyl group fills the site occupied by the phenyl group of Phe56 in the wild-type complex. In contrast, an extended conformation of A-4CPh is predicted to be most stable in the complex formed with the wild-type protein. The calculations indicate A-4CPh is in an extended conformation in the free RNA. Therefore, preorganizing the structure of the phenyl-tethered base for binding may improve both the affinity and specificity of the RNA containing A-4CPh for the Phe56Ala U1A protein. Taken together, the previous experimental work and the calculations reported here suggest a general design strategy for altering RRM-RNA complex stability.     

Multiple phosphorylation sites of DNA polymerase alpha-primase cooperate to regulate the initiation of DNA replication in vitro

DNA polymerase alpha-primase (pol-prim) is the only enzyme that can start DNA replication de novo. The 180-kDa (p180) and 68-kDa (p68) subunits of the human four-subunit enzyme are phosphorylated by Cyclin-dependent kinases (Cdks) in a cell cycle-dependent manner. Cyclin A-Cdk2 physically interacts with pol-prim and phosphorylates N-terminal amino acids of the p180 and the p68 subunits, leading to an inhibition of pol-prim in initiating cell-free SV40 DNA replication. Mutation of conserved putative Cdk phosphorylation sites in the N terminus of human p180 and p68 reduced their phosphorylation by Cyclin A-Cdk2 in vitro. In contrast to wild-type pol-prim these mutants were no longer inhibited by Cyclin A-Cdk2 in the initiation of viral DNA replication. Importantly, rather than inhibiting it, Cyclin A-Cdk2 stimulated the initiation activity of pol-prim containing a triple N-terminal alanine mutant of the p180 subunit. Together these results suggest that Cyclin A-Cdk2 executes both stimulatory and inhibitory effects on the activity of pol-prim in initiating DNA replication.     

Relationships between heme incorporation, tetramer formation, and catalysis of a heme-regulated phosphodiesterase from Escherichia coli: a study of deletion and site-directed mutants

The heme-regulated phosphodiesterase (PDE) from Escherichia coli (Ec DOS) is a tetrameric protein composed of an N-terminal sensor domain (amino acids 1-201) containing two PAS domains (PAS-A, amino acids 21-84, and PAS-B, amino acids 144-201) and a C-terminal catalytic domain (amino acids 336-799). Heme is bound to the PAS-A domain, and the redox state of the heme iron regulates PDE activity. In our experiments, a H77A mutation and deletion of the PAS-B domain resulted in the loss of heme binding affinity to PAS-A. However, both mutant proteins were still tetrameric and more active than the full-length wild-type enzyme (140% activity compared with full-length wild type), suggesting that heme binding is not essential for catalysis. An N-terminal truncated mutant (DeltaN147, amino acids 148-807) containing no PAS-A domain or heme displayed 160% activity compared with full-length wild-type protein, confirming that the heme-bound PAS-A domain is not required for catalytic activity. An analysis of C-terminal truncated mutants led to mapping of the regions responsible for tetramer formation and revealed PDE activity in tetrameric proteins only. Mutations at a putative metal-ion binding site (His-590, His-594) totally abolished PDE activity, suggesting that binding of Mg2+ to the site is essential for catalysis. Interestingly, the addition of the isolated PAS-A domain in the Fe2+ form to the full-length wild-type protein markedly enhanced PDE activity (>5-fold). This activation is probably because of structural changes in the catalytic site as a result of interactions between the isolated PAS-A domain and that of the holoenzyme.     

巨大芽孢杆菌b-淀粉酶基因的克隆、表达和酶学性质分析

从巨大芽孢杆菌(Bacillus megaterium)的全基因组DNA文库中筛选出一个b-淀粉酶基因amyG, 分析测定了其核苷酸序列并进行了诱导表达; 其中amyG编码的蛋白有545个氨基酸、分子量为60.194 kD, 与已报道的巨大芽孢杆菌DSM319的b-淀粉酶序列有着94.5%的同源性。经氨基酸序列比较分析发现, AmyG从N末端到C末端依次由信号肽域、糖基水解酶催化功能域和淀粉结合域3个功能域组成。其中催化功能域里含有第14家族糖基水解酶常见的几个高度保守的酶催化活性区。经多步纯化, 重组酶的比活共提高了7.4倍, 获得凝胶电泳均一的蛋白样品; 经SDS-PAGE电泳测定, 酶AmyG的分子量为57 kD。该酶的最适反应温度为60oC, 最适反应pH为7.0; 在温度不超过60oC时, 酶活较稳定; AmyG能迅速降解淀粉生成麦芽糖, 属于外切b-糖苷酶。     

Biochemical and genetic analyses of a novel gamma-cyclodextrin glucanotransferase from an alkalophilic Bacillus clarkii 7364

On screening for microorganisms in soil obtained in Japan that produce large amounts of gamma-cyclodextrin (gamma-CD), we identified a novel alkalophilic bacterium, Bacillus clarkii 7364. The cyclodextrin glucanotransferase (CGTase) secreted into the culture medium by this bacterium was purified by affinity chromatography on a gamma-CD-immobilized column, followed by chromatography on a gel filtration column. The enzyme converted 13.7% of pre-gelatinized potato starch (10% w/w per reaction mixture) into CDs, and the majority (79%) of the product CDs was of the gamma form. This property is quite unique among known CGTases and thus we named this enzyme gamma-CGTase. The N-terminal and internal amino acid sequences of gamma-CGTase were determined and used to design PCR primers for amplification of the nucleotide sequence that encodes the gamma-CGTase gene. The entire gene sequence amplified by PCR was determined and then cloned into E. coli. The recombinant enzyme synthesized by E. coli retained biochemical properties quite similar to those of the original one. Comparison of the deduced amino acid sequence of gamma-CGTase with those of other known CGTases that have different product specificities revealed the importance of subsites -3 and -7 for the preferential gamma-cyclization activity.     

Expression of the transglutaminase gene in Escherichia coli.

1. The cDNA gene coding for the enzyme transglutiminase (EC 2.3.2.13) was cloned into the pUC18 oriented for expression from the lac promoter. 2. DNA sequencing of the 5' end showed that the cDNA was missing the sequence coding of the N-terminal 30 amino acids. 3. The truncated gene was then cloned into pKK233-2, and the recombinant product was produced in Escherichia coli. 4. A gene construct coding for the complete protein was generated by inserting an oligonucleotide for the missing 30 amino acids into the Eco RI site of the pUC18 clone. 5. A consensus Shine-Dalgarno sequence and translational start codon were positioned at the 5' end of the linker. 6. Immunoblotting experiments of E. coli JM105(pUC18-TGase) indicated the expression of the transglutaminase gene. 7. The cell lysate as well as the partially purified transglutaminase showed no detectable enzyme activity.     

Proteinase Enzyme System of Lactic Streptococci III. Substrate Specificity of Streptococcus lactis Intracellular Proteinase

The substrate specificity of an intracellular proteinase from Streptococcus lactis was investigated in an effort to understand the role of the enzyme in the cell. Peptides in which the N-terminal residue was glycine were not hydrolyzed by the enzyme (exceptions were glycyl-alanine, glycyl-aspartic acid, and glycyl-asparagine), but the peptide was hydrolyzed if the N-terminal residue was alanine. The enzyme also showed activity toward peptides containing aspartic acid or asparagine. Hydrolysis of only the peptide bonds of alanyl, aspartyl, or asparaginyl residues was confirmed by the action of the enzyme on oxidized bovine ribonuclease A- and B- chain insulin. The N-terminal residues of the peptide fragments liberated were identified. The enzyme attacked both substrates only at alanyl, aspartyl, and asparaginyl residues, releasing these as free amino acids. In addition to alanine, aspartic acid, and asparagine, certain other amino acids were liberated from ribonuclease A, but these were accounted for by the relation of their position to alanine, aspartic acid, and asparagine residues.