A survey of furin substrate specificity using substrate phage display.

The substrate specificity of furin, a mammalian enzyme involved in the cleavage of many constitutively expressed protein precursors, was studied using substrate phage display. In this method, a multitude of substrate sequences are displayed as fusion proteins on filamentous phage particles and ones that are cleaved can be purified by affinity chromatography. The cleaved phage are propagated and submitted to additional rounds of protease selection to further enrich for good substrates. DNA sequencing of the cleaved phage is used to identify the substrate sequence. After 6 rounds of sorting a substrate phage library comprising 5 randomized amino acids (xxxxx), virtually all clones had an RxxR motif and many had Lys, Arg, or Pro before the second Arg. Nine of the selected sequences were assayed using a substrate-alkaline phosphatase fusion protein system. All were cleaved after the RxxR, and some substrates with Pro or Thr in P2 were also found to be cleaved as efficiently as RxKR or RxRR. To further elaborate surrounding determinants, we constructed 2 secondary libraries (xxRx(K/R)Rx and xxRxPRx). Although no consensus developed for the latter library, many of the sequences in the the former library had the 7-residue motif (L/P)RRF(K/R)RP, suggesting that the furin recognition sequence may extend over more than 4 residues. These studies further clarify the substrate specificity of furin and suggest the substrate phage method may be useful for identifying consensus substrate motifs in other protein processing enzymes.     

Purification of human renin substrate.

Renin substrate, angiotensinogen, has been purified from human plasma by methods which permit the processing of large amounts of outdated bank blood. The purified protein is homogeneous by disc gel electrophoresis at pH 9.5. The specific activity of 18 nmol/mg corresponds to a molecular weight of 56,000, while a higher value, 90,000, is found by gel filtration. Chromatography of partially purified renin substrate on DEAE-cellulose in a descending pH gradient shows evidence for the existence of multiple forms. However, some of these forms appear to be lost after chromatography on hydroxylapatite.     

Donor substrate regulation of transketolase.

The influence of substrates on the interaction of apotransketolase with thiamin diphosphate was investigated in the presence of magnesium ions. It was shown that the donor substrates, but not the acceptor substrates, enhance the affinity of the coenzyme either to only one active center of transketolase or to both active centers, but to different degrees in each, resulting in a negative cooperativity for coenzyme binding. In the absence of donor substrate, negative cooperativity is not observed. The donor substrate did not affect the interaction of the apoenzyme with the inactive coenzyme analogue, N3'-pyridyl-thiamin diphosphate. The influence of the donor substrate on the coenzyme-apotransketolase interaction was predicted as a result of formation of the transketolase reaction intermediate 2-(alpha,beta-dihydroxyethyl)-thiamin diphosphate, which exhibited a higher affinity to the enzyme than thiamin diphosphate. The enhancement of thiamin diphosphate's affinity to apotransketolase in the presence of donor substrate is probably one of the mechanisms underlying the substrate-affected transketolase regulation at low coenzyme concentrations.     

Hypersensitive substrate for ribonucleases.

A substrate for a hypersensitive assay of ribonucleolytic activity was developed in a systematic manner. This substrate is based on the fluorescence quenching of fluorescein held in proximity to rhodamine by a single ribonucleotide embedded within a series of deoxynucleotides. When the substrate is cleaved, the fluorescence of fluorescein is manifested. The optimal substrate is a tetranucleotide with a 5',6-carboxyfluorescein label (6-FAM) and a 3',6-carboxy-tetramethylrhodamine (6-TAMRA) label: 6-FAM-dArUdAdA-6-TAMRA. The fluorescence of this substrate increases 180-fold upon cleavage. Bovine pancreatic ribonuclease A (RNase A) cleaves this substrate with a k (cat)/ K (m)of 3.6 x 10(7)M(-1)s(-1). Human angiogenin, which is a homolog of RNase A that promotes neovascularization, cleaves this substrate with a k (cat)/ K (m)of 3. 3 x 10(2)M(-1)s(-1). This value is >10-fold larger than that for other known substrates of angio-genin. With these attributes, 6-FAM-dArUdAdA-6-TAMRA is the most sensitive known substrate for detecting ribo-nucleolytic activity. This high sensitivity enables a simple protocol for the rapid determination of the inhibition constant ( K (i)) for competitive inhibitors such as uridine 3'-phosphate and adenosine 5'-diphos-phate.     

Enzyme substrate and inhibitor interactions.

An enzyme is designed to bind most tightly to a substrate when it is in the transition state of the reaction which the enzyme catalyses. The consequent reduction of the activation energy of the reaction constitutes the catalytic mechanism. The energetic contributions of different features of the interaction can only be crudely assessed, but they are dominated by entropically driven effects. The binding site of trypsin orients the substrate so that the reacting groups are correctly placed for reaction to occur. Apart from two side chains which take part in chemical steps of the reaction, the enzyme behaves almost as a rigid body. The full binding interactions are only developed when the substrate is in an intermediate stage of the reaction. The tightly bound complexes of trypsin with protein trypsin inhibitors have proved amenable to structural analysis. Enzyme inhibitor interactions, which account for almost 80 kJ mol-1 of interaction energy, are known fairly accurately. The similarity of the two known trypsin inhibitor structures, close to the primary binding site, indicates a high specificity, even for this simple interaction. In cases where no large conformational changes occur the specificity of an enzyme should be predictable from accurate knowledge of its tertiary structure.     

Reversible inhibition in bimolecular rapid-equilibrium random-order enzyme systems. The effect of substrate–substrate and inhibitor–substrate interactions

A model is presented that accounts for all types of reversible inhibition by a single inhibitor molecule in bimolecular rapid-equilibrium random-order enzyme systems. The characterization of inhibition mechanisms by graphical methods is examined, and a system of nomenclature is suggested.     

The crystal structure of a phosphorylase kinase peptide substrate complex: kinase substrate recognition.

The structure of a truncated form of the gamma-subunit of phosphorylase kinase (PHKgammat) has been solved in a ternary complex with a non-hydrolysable ATP analogue (adenylyl imidodiphosphate, AMPPNP) and a heptapeptide substrate related in sequence to both the natural substrate and to the optimal peptide substrate. Kinetic characterization of the phosphotransfer reaction confirms the peptide to be a good substrate, and the structure allows identification of key features responsible for its high affinity. Unexpectedly, the substrate peptide forms a short anti-parallel beta-sheet with the kinase activation segment, the region which in other kinases plays an important role in regulation of enzyme activity. This anchoring of the main chain of the substrate peptide at a fixed distance from the gamma-phosphate of ATP explains the selectivity of PHK for serine/threonine over tyrosine as a substrate. The catalytic core of PHK exists as a dimer in crystals of the ternary complex, and the relevance of this phenomenon to its in vivo recognition of dimeric glycogen phosphorylase b is considered.     

Synthesis of renin substrate by rat liver.

The present study attempts to determine if the isolated rat liver is capable of synthesizing renin substrate from 14C-labelled amino acids added in the perfusate. The renin substrate is characterized via reaction with renin, forming a substance that is subsequently identified as proangiotensin. Extensive evaluation of the reaction product is carried out by using molecular-sieve chromatography, countercurrent distribution, reactivity with converting enzyme, radioimmunological technique and bioassay. The results demonstrate that isolated rat liver perfused with artificial salt solution is capable of synthesizing a protein that reacts with renin to form a radioactive substance indistinguishable from proangiotensin.     

Violaxanthin de-epoxidase. Lipid composition and substrate specificity.

Violaxanthin de-epoxidase isolated from lettuce chloroplasts (Lactuca sativa var. Romaine) contained a single lipid component, monogalactosyldiglyceride (MG) at about 8 g per 100 g protein. The effects of MG on activation of solvent-extracted enzyme and on K_m suggest that MG has two roles, namely, as a functional component of the binding site and as a substrate-solubilizing agent whose structure satisfies binding site requirements. Substrate specificity examined with various naturally occurring and semisynthetic epoxy carotenoids with known chirality showed violaxanthin de-epoxidase to be stereospecific for 3-hydroxy, 5,6-epoxy carotenoids which are in a 3S, 5R, 6S configuration. Although monoepoxides with the above configuration were active, their rates varied, apparently due to the influence of structural differences in the nonepoxide end groups. Hence while all-trans neoxanthin showed low rates, the de-epoxidation rate of antheraxanthin was 5-fold higher than violaxanthin. Neoxanthin and violeoxanthin, both naturally occurring pigments with 9-cis configurations in the acyclic polyene chain, were inactive. These effects support the view that violaxanthin de-epoxidase is a mono de-epoxidase and that the stereospecific active center is situated in a narrow well-like cavity which favors an all-trans configuration of the polyene chain. The 3-hydroxy, 5,6-epoxy group of the naturally occurring pigments, diadinoxanthin, antheraxanthin, and β-cryptoxanthin epoxide are assumed to be the 3S, 5R, 6S configuration based on their reactivity with violaxanthin de-epoxidase.     

Characterization and substrate specificity of fumarylacetoacetate fumarylhydrolase.

The molecular weight of fumarylacetoacetate fumarylhydrolase (EC 3.7.1.2) is 86 000 +/- 10 000, as determined by gel filtration. The enzyme appears to be a dimer with a monomer molecular weight of 38 000 - 43 000, as determined by gel electrophoresis, gel filtration in guanidine-hydrochloride, and ultracentrifugation. The subunits appear to be identical, as only one band is seen in gel electrophoresis, only one protein peak is detected in gel filtration in guanidine-hydrochloride, and only one amino-terminal amino acid (proline) is detected. Three free sulfhydryl groups per denatured monomer are detected by reaction with 5,5'-dithiobis(2-nitrobenzoic acid), while for the active enzyme only two sulfhydryl groups react with this reagent, The extinction coefficients at 260 and 280 nm, the amino acid composition, and the isoelectric point (6.7) of the enzyme are also reported. The enzyme catalyzes the hydrolysis of six 2,4-diketo acids and three 3,5-diketo acids tested. The Km of the substrates is similar but V varies by a factor of 120. The pH optimum is 7.3. The enzyme did not catalyze the hydrolysis of a number of esters tested.     

Electrostatic reversal of serine proteinase substrate specificity.

Mouse granzyme B is a member of the chymotrypsin family of serine proteinases that has an unusual preference for cleavage of substrates following aspartate residues. We show here that granzyme B can be redesigned by a single amino acid substitution in one wall of the specificity pocket, arginine-226 to glutamate, to hydrolyze preferentially thioester substrates following basic amino acids. Amide substrates, however, were not hydrolyzed by the variant granzyme B. These results show that residue 226 is a primary determinant of granzyme B specificity and imply that additional structural components are required for catalysis of amide bonds. Molecular modeling indicated subtle variation in glutamate-226 orientation depending upon the state of protonation of the gamma-carboxylate, which may account for the secondary specificity of this enzyme for substrates containing phenylalanine. This represents the first example of electrostatic reversal of serine proteinase substrate specificity and suggests that residue 226 is a primary substrate specificity determinant in the granzyme B lineage of serine proteinases.