James C. Powers

Reaction of acyl carbazates with proteolytic enzymes

Abstract Acyl carbazates (1) with appropriate substituents are analogs of acyl amino acids in which the α-methine group has been replaced by a nitrogen atom. The synthesis of three new carbazates Ac-Ala-Bzc-ONp, Ac-Ala-Mec-ONp and Z-Ala-Ala-Pro-Mec-ONp (Bzc= ue5f8NHN(CH 2 C 6 H 5 )COue5f8, Mec= ue5f8NHN(CH 3 )COue5f8) was accomplished. The reaction of chymotrypsin, subtilisin BPN′, and elastase (procine and human leukocyte) with the carbazates resulted either in a spectrophotometric burst of p -nitrophenol or no reaction depending on the specificity of the enzyme. Carbazyl chymotrypsin A α crystals were isomorphous with the native protein. Acyl carbazates should find utility as inhibitors and active site titrants for serine proteases.

Inhibition of human leukocyte elastase by peptide chloromethyl ketones

Recently, an elastolytic protease has been isolated from human polymorphonuclear (PMN) leukocyte granule fractions and purified by affinity chromatography [ 11. This human leukocyte elastase is known to degrade human lung elastin, arterial walls and basement membrane, digest proteins of bacterial cell walls in vitro, induce cellular surface changes correlated with loss of growth control, and undergo inhibition by cyI antitrypsin and peptide chloromethyl ketones [2]. A growing body of experimental evidence has suggested that this elastase may be involved in pathological processes associated with elastic tissue damage in pulmonary emphysema and acute arteritis, and with the tumor-producing action of cocarcinogenic substances [3]. Peptide chloromethyl ketones are well-known inhibitors of serine proteases which have proved quite useful for structural studies, including X-ray crystallographic investigations, of the active site regions of this group of enzymes [4]. A series of alanine triand tetrapeptide chloromethyl ketone inhibitors was synthesized by us and two other groups to probe the nature of the extended binding site and catalytic activity of porcine pancreatic elastase (EC 3.4.2 1 .l 1) and related enzymes [5,6 1. In this paper we report a study of the inhibition of human leukocyte elastase by a series of peptide chloromethyl ketones, which demonstrates that the human leukocyte enzyme is distinct from the porcine pancreatic enzyme. The results also define the structural features necessary for an effective inhibitor and may lead to a treatment for emphysema and related diseases.

PROTEOLYTIC EVENTS IN REPLICATION OF ANIMAL VIRUSES

It has been clear for more than 10 years that proteolytic modifications of viral proteins occur during replication (reviewed in reference 1). Most commonly observed are cleavages of structural protein precursors, which cause profound modifications in the surface properties of the products, and lead to their aggregation into specific supermolecular structures with molecular weights of millions, and abilities to combine with viral nucleic acids and to recognize cellular surface receptors. With many of the viruses proteolytic cleavages occur during protein biosynthesis or just after translation is completed. The roles of these cleavages are virtually unknown. Until recently, the origin of the proteolytic enzymes that process the viral precursors was vague; however recent advances in studies of bacteriophages,* RNA tumor viru~es,~ togaviruses,‘ and picornaviru~e~~” have provided evidence for virus-coded proteases involved in the ultimate stages of processing. In this study we used precursors of picornavirus proteins to analyze the contribution of cellular and virusspecific proteases in the processing reactions.

SYNTHETIC ACTIVE SITE‐DIRECTED INHIBITORS OF ELASTOLYTIC PROTEASES*

Several proteolytic enzymes are known to occur in human polymorphonuclear (PMN) leukocyte granule fractions, including an elastase that is active at physiologic pH.l-* The human leukocyte elastase has been isolated and purified by affinity chromatography. It is known to degrade human lung elastin, arterial walls, and basement membrane, digest proteins of bacterial cell walls in vitro, induce cellular surface changes correlated with !oss of growth control, and undergo inhibition by a,-antitrypsin and peptide chloromethyl ketone^.^-^ A growing body of experimental evidence has suggested that this elastase may be involved in pathologic processes associated with elastic tissue damage in acute arthritis, pulmonary emphysema, and related diseases.l0-l3 Thus, synthetic elastase inhibitors would be expected to be useful reagents both for the treatment of arthritis, emphysema, and related diseases and for the study of the biologic function of elastolytic enzymes. Human leukocyte elastase is similar to the more widely studied porcine pancreatic elastase l4 in many respects. They are both serine proteases and show esterase activity toward synthetic substrates, such as Boc-Ala-ONp and Ac-Ala-Ala-Ala-OCH,. One very useful class of inhibitors for serine proteases is the chloromethyl ketones. These inhibitors, if properly designed to resemble a substrate, are fairly specific for one serine protease or a group of serine proteases with similar specificities. The reason for this specificity is apparent upon consideration of the mechanism of action of serine proteases. A substrate is bound in such a way that the carbonyl group of the scissile peptide bond is properly oriented to be attacked by the active-site serine residue, which action leads to an acyl enzyme intermediate that rapidly turns over (FIGURE 1 ) . A similarly bound chloromethyl ketone inhibitor (FIGURE 1 ), however, would place the active-site histidine residue next to the alkylating group of the inhibitor, which results in the formation of a covalent bond between enzyme and inhibitor. Attack by the active-site serine residue on the carbonyl group of the inhibitor would lead to no stable products. A series of aianine triand tetrapeptide chloromethyl ketones was recently synthesized by us l 7 and subsequently by two independent groups 18, In as elastase inhibitors. The inhibitors were designed to contain two features thought to be essential for effective inhibition, a terminal alanine chloromethyl ketone moiety due to the known specificity of elastase and an extended peptide chain, because the rate of elastase hydrolysis of synthetic substrates is strongly