Kinetics of the inhibition of fusarium serine proteinases by barley (Hordeum vulgare L.) inhibitors.

Fungal infections of barley and wheat cause devastating losses of these food crops. The endogenous proteinase inhibitors produced by plant seeds probably defend the plants from pathogens by inhibiting the degradation of their proteins by the pathogen proteases. We have studied the interactions of barley grain inhibitors with the subtilisin-like and trypsinlike proteinases of Fusarium culmorum. The inhibition kinetics of three inhibitor proteins, chymotrypsin/subtilisin inhibitor 2 (CI-2), barley alpha-amylase/subtilisin inhibitor (BASI), and Bowman-Birk trypsin inhibitor (BBBI), have been studied in detail for the first time using fungal enzymes. The kinetic studies were performed at physiological pH values to mimic in vivo conditions. Numerical approaches to kinetic analyses were used to calculate the inhibition constants, because the data analyses were complicated by some inhibitor turnover and the instability of enzymes and substrates. All were slow, tight-binding inhibitors that followed either a two-step mechanism (CI-2 and BASI) or a single-step mechanism (BBBI) under the conditions investigated. The overall Ki values derived were approximately 50 pM, 1 nM, and 0.1 nM for CI-2, BASI, and BBBI, respectively. The main difference between the CI-2 and the BASI inhibitions was accounted for by the stabilities of their final complexes and the rate constants for their second dissociation steps (9 x 10(-6)/s and 3 x 10(-4)/s, respectively). Understanding the inhibition mechanisms will be valuable in designing improved strategies for increasing the resistance of the grains to fungal infections.

Effect of reducing and oxidizing agents and pH on malt endoproteolytic activities and brewing mashes.

The activities of the four endoproteinase classes of malted barley are known to vary with pH, and it seemed likely that the cysteine enzyme activities could be altered by redox agents. This study determined how altering the pH and adding redox agents to mashes influenced the worts that were produced during the brewing process. The reducing agents cysteine.HCl, dithiothreitol, and beta-mercaptoethanol increased the proteolysis that occurred in malt extracts and mashes. This increased proteolysis was negated by the addition of the oxidizing agents diamide or hydrogen peroxide. The addition of reducing agents to mashes increased the soluble protein, free amino nitrogen (FAN), and extract values of their resultant worts, and this effect was abolished by the concomitant addition of oxidizing agents. Raising the pH values of the mashes strongly reduced their proteolytic activities, soluble protein, FAN, and extract values, but not their beta-glucan levels. These results show that several of the major aspects of malting and brewing quality can be adjusted by varying the pH and redox qualitites of mashes, which could be helpful to brewers. These results also strengthen the previous proposal made by Buchanan et al. that the redox status of plants may play a significant part in controlling their physiology.

Trypsin/alpha-amylase inhibitors inactivate the endogenous barley/malt serine endoproteinase SEP-1.

Barley (Hordeum vulgare L.) malt contains endoproteinases belonging to all four of the commonly occurring classes, including serine proteinases. It also contains low molecular weight proteins that inhibit the activities of many of these endoproteinases, but it had never been shown that any barley or malt serine proteinases could be inhibited by any of these endogenous proteins. It is now reported that some proteins that were concentrated using an "affinity" method inhibited the activity of a malt serine endoproteinase. Two-dimensional electrophoretic and in vitro analyses showed that the inhibited enzyme was serine endoproteinase 1 (SEP-1) and that the inhibition could be quantified using a semipurified preparation of this enzyme. Amino acid sequencing and MALDI-TOF MS were used to identify the components of the partially purified inhibiting fractions. Only the "trypsin/alpha-amylase inhibitors" or chloroform/methanol (CM) proteins, most of which had truncated N and C termini, and one fragment of beta-amylase were present in the inhibitory fractions. When a CM protein fraction was prepared from barley according to traditional methods, some of its component proteins inhibited the activity of SEP-1 and some did not. This is the first report of the purification and identification of barley malt proteins that can inhibit an endogenous serine proteinase. It shows that some of the CM proteins probably play a role in controlling the activity of barley proteinases during germination, as well as possibly protecting the seed and young plant from microbes or pests.

Purification and identification of barley (Hordeum vulgare L.) proteins that inhibit the alkaline serine proteinases of Fusarium culmorum.

It has been proposed that microbial proteinase inhibitors, which are present in abundance in cereal grains, protect the seed against plant pathogens. So far, however, very little is known about the interactions of those inhibitors with the proteinases of phytopathogenic microbes. The increased alkaline proteinase activities of Fusarium head blight (FHB) diseased wheat and barley grain imply that the Fusarium fungi synthesize those enzymes during the colonization of the kernel. To study which barley proteins can inhibit Fusarium proteinases, and hence, possibly protect the seed from FHB, the proteins of a grain extract have been separated and tested for their abilities to inhibit two alkaline serine proteinases that we previously isolated from F. culmorum. The proteins were separated by size exclusion, ion exchange, and reversed-phase-HPLC chromatographies. The purified inhibitors were identified by their molecular masses and N-terminal amino acid sequences. The proteins that inhibited the subtilisin-like Fusarium proteinase were the chymotrypsin/subtilisin (CI) inhibitors 1A, 1B, and 2A and the barley alpha-amylase/subtilisin inhibitor (BASI). Only one of the purified proteins inhibited the trypsin-like proteinase, the barley Bowman-Birk inhibitor (BBBI). No novel inhibitors were detected.

SEP-1 - a subtilisin-like serine endopeptidase from germinated seeds of Hordeum vulgare L. cv. Morex.

Proteolysis is crucial for all living cells. It regulates protein processing, intracellular protein levels and removes abnormal or damaged proteins from the cell, working as a cellular housekeeper. By means of proteolysis, cells can control the short-lived regulatory proteins that affect processes such as signal transduction and reception, transcription, division and cellular growth. Proteolysis also furnishes amino acids for the de novo synthesis of proteins. In germinating seeds, its main role is to degrade storage proteins into small peptides and amino acids that can be used by the embryo during autotrophic growth. We have isolated and purified a serine endopeptidase, one of the many proteolytic enzymes that occur in germinated barley seeds (green malt), using chromatofocusing and DEAE-, CM-, and size-exclusion chromatographies. The enzyme, named SEP-1, has a molecular weight of 70 kDa, as estimated by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography. SEP-1 was detected and measured by its ability to digest gelatin in gels and to hydrolyze the synthetic substrate N-succinyl Ala-Ala-Pro-Leu p-nitroanilide. The hydrolysis of the synthetic substrate was optimal at pH 6.5 and 50 degrees C with a K(m) of 2.6 mM. The enzyme was inhibited by phenylmethylsulfonyl fluoride and p-amidinophenyl methanesulfonyl fluoride but not by any other class-specific inhibitor, suggesting it was a serine endopeptidase. Its amino acid sequence was similar to those of other plant subtilisin-like serine peptidases (EC 3.4.21), especially to the cucumisin-like group. SEP-1 was present in resting seeds, and its activity increased during germination in all of the malted barley tissues except for the endosperm, where it never occurred, suggesting that the enzyme is not likely involved in storage-protein degradation.

Trypsin-like proteinase produced by Fusarium culmorum grown on grain proteins.

The fungal disease Fusarium head blight occurs on wheat (Triticum spp.) and barley (Hordeum vulgare L.) and is one of the worldwide problems of agriculture. It can be caused by various Fusarium species. We are characterizing the proteinases of F. culmorum to investigate how they may help the fungus to attack the grain. A trypsin-like proteinase has been purified from a gluten-containing culture medium of F. culmorum. The enzyme was maximally active at about pH 9 and 45 degrees C, but was not stable under those conditions. It was stabilized by calcium ions and by the presence of other proteins. The proteinase was most stable at pH 6-7 at ambient temperatures, but was quickly inactivated at 50 degrees C. It was strongly inhibited by p-amidino phenylmethylsulfonyl fluoride (p-APMSF), and soybean trypsin and Bowman-Birk inhibitors, and it preferentially hydrolyzed the peptide bonds of the protein substrate beta-purothionin on the C-terminal side of Arg (mainly) and Lys residues. These characteristics show that it is a trypsin-like proteinase. In addition, its N-terminal amino acid sequence was 88% identical to that of the F. oxysporum trypsin-like enzyme. The proteinase hydrolyzed the D hordein and some of the C hordeins (the barley storage proteins). This enzyme, and a subtilisin-like proteinase that we recently purified from the same organism, possibly play roles in helping the fungus to colonize grains.

Purification and properties of an alkaline proteinase of Fusarium culmorum.

The disease Fusarium head blight (scab) causes severe problems for farmers and for the industries that use cereals. It is likely that the fungi that cause scab (Fusarium spp.) use various enzymes when they invade grains. We are studying enzymes that the fungi may use to hydrolyze grain proteins. To do this, Fusarium culmorum was grown in a gluten-containing medium from which an alkaline serine proteinase with a molecular mass of 28.7 kDa was purified by size-exclusion and cation exchange chromatographies. The enzyme was maximally active at pH 8.3-9.6 and 50 degrees C, but was unstable under these conditions. It hydrolyzed the synthetic substrates N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide and, to a lesser extent, N-succinyl-Ala-Ala-Pro-Leu p-nitroanilide. It was inhibited by phenylmethanesulfonyl fluoride and chymostatin, but not by soybean trypsin or Bowman-Birk inhibitors. Parts of the amino-acid sequence were up to 82% homologous with those of several fungal subtilisins. One of the active site amino acids was detected and it occupied the same relative position as in the other subtilisins. Therefore, on the basis of these characteristics, the proteinase is subtilisin-like. Purification of the enzyme was complicated by the fact that, when purified, it apparently underwent autolysis. The presence of extraneous protein stabilized the activity.

The effect of mashing on malt endoproteolytic activities.

During malting and mashing, the proteinases of barley (Hordeum vulgare L.) and malt partially hydrolyze their storage proteins. These enzymes are critical because several aspects of the brewing process are affected by the soluble proteins, peptides and/or amino acids that they release. To develop improved malting barleys and/or malting and brewing methods, it is imperative to know whether and when the green malt endoproteinases are inactivated during malting and mashing. These enzyme activities are totally preserved during kilning and, in this study, we have determined when they were inactivated during mashing. Samples were removed from experimental mashes that mirrored those used in commercial breweries and their endoproteolytic activities were analyzed. The malt endoproteinases were stable through the 38 degrees C protein rest phase, but were quickly inactivated when the mash temperature was raised to 72 degrees C for the conversion step. All of the proteinase activities were inactivated at about the same rate. These findings indicate that the soluble protein levels of worts can be varied by adjusting the protein rest phase of mashing, but not by altering the conversion time. The rates of hydrolysis of individual malt proteins probably cannot be changed by altering the mash temperature schedule, since the main enzymes that solubilize these proteins are affected similarly by temperature.